Methods and compounds for modifying mitochondrial function

Abstract

The present invention provides methods of identifying mitochondrial function regulators for use in the treatment of neurodegenerative disorders. The invention provides methods for identifying subjects that can benefit from the therapeutic agent. Aspects of the methods include administering a MIRO1 reducing agent to a subject suffering from Parkinson's disease. The invention also provides companion diagnostic assays to determine whether a subject is suitable for receiving MIRO1 reducing agent treatment and treat the subject according to the results.

Description

[0001] Division Application Description

[0002] This patent application is a divisional application of the Chinese patent application with the application number 2020800766141 and the title "Methods and Compounds for Modifying Mitochondrial Function", which was filed on September 4, 2020.

[0003] Cross - reference to Related Patent Applications

[0004] This patent application claims the priority of the US Provisional Patent Application No. 62 / 896,450, filed on September 5, 2019, the entire text of which is incorporated herein by reference for all purposes. Technical Field

[0005] This application relates to therapeutic means and methods for neurodegenerative disorders. In particular, it relates to methods and compounds for modifying mitochondrial function. Background Art

[0006] Neurons are metabolically active cells with high energy demands at locations far from the cell body. Thus, these cells are particularly dependent on mitochondrial function, as reflected by the observation that mitochondrial dysfunction diseases often involve neurodegenerative disorders. Recent findings indicate that neurons are particularly dependent on the dynamic properties of mitochondria. By several criteria, mitochondria are dynamic organelles. They participate in repetitive fusion and fission cycles, which helps to mix lipids and contents in the mitochondrial population. In addition, mitochondria are actively recruited to sub - cellular sites, such as during the development of axons and dendrites of neurons. Finally, the quality of the mitochondrial population is maintained by mitophagy, a form of autophagy that selectively degrades defective mitochondria. Defects in key aspects of mitochondrial dynamics (such as mitochondrial fusion, fission, transport, and mitophagy) are associated with neurodegenerative disorders. Multiple major neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, and Huntington's disease, involve disruptions to mitochondrial dynamics.

[0007] Mitochondrial movement is tightly controlled to maintain energy homeostasis and prevent oxidative stress. Mitochondrial movement stops prior to the onset of mitophagy, an important cellular mechanism by which depolarized mitochondria are degraded by autophagosomes and lysosomes. Movement arrest can sequester damaged mitochondria, preventing their movement and simultaneously preventing the re - introduction of damage to other healthy mitochondria.

[0008] Miro is an outer mitochondrial membrane (OMM) protein that anchors the microtubule motor proteins, kinesin and dynein, to mitochondria (Glater EE, Megeath LJ, Stowers RS, Schwarz TL. Axonal transport of mitochondria requires Milton to recruit kinesin heavy chain and is independent of the light chain. Journal of Cell Biology. 2006;173:545–557; Koutsopoulos OS, Laine D, Osellame L, Chudakov DM, Parton RG, Frazier AE, Ryan MT. Human Milton associates with mitochondria and induces microtubule-dependent remodeling of the mitochondrial network. Biochimica et Biophysica Acta. 2010;1803:564–574.). Removal of Miro from the surface of damaged mitochondria enables this depolarization-triggered mitochondrial arrest (Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D, Rice S, Steen J, LaVoie MJ, Schwarz TL. PINK1 and Parkin target Miro for phosphorylation and degradation to halt mitochondrial motility. Cell. 2011;147:893-906.). Miro is subsequently degraded by the proteasome (Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D, Rice S, Steen J, LaVoie MJ, Schwarz TL. PINK1 and Parkin target Miro for phosphorylation and degradation to halt mitochondrial motility. Cell. 2011;147:893-906.). There is evidence that two PD-related proteins, PINK1 (PTEN-induced putative kinase 1) and Parkin, act in concert to target Miro for degradation (Ashrafi G, Schlehe JS, LaVoie MJ, Schwarz TL. Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin. Journal of Cell Biology. 2014;206:655–670; Liu S, Sawada T, Lee S, Yu W, Silverio G, Alapatt P, Millan I, Shen A, Saxton W, Kanao T et al. The Parkinson's disease-related kinase PINK1 regulates Miro protein levels and axonal transport of mitochondria. PLoS Genetics.2012; 8: e1002537; Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D, Rice S, Steen J, LaVoie MJ, Schwarz TL. PINK1 and Parkin target Miro for phosphorylation and degradation to prevent mitochondrial motility. Cell. 2011; 147: 893-906.). Mutations in PINK1 or Parkin are associated with rare recessive early-onset PD.

[0009] Altered mitochondrial trafficking is one of the pathogenic changes in major adult-onset neurodegenerative diseases. In mutant LRRK2GS2019 cells, the mitochondrial outer membrane protein Miro remains stable and stays on damaged mitochondria longer than on normal mitochondria, which results in prolonged active transport and inhibited mitochondrial degradation (Hsieh et al., 2016). Miro degradation and mitochondrial motility are also adversely affected in subjects with sporadic PD. The prolonged residence of Miro and the consequent downstream consequences may be a core component of the PD pathogenesis.

[0010] Therefore, new therapeutic means and methods are needed to identify regulators of Miro retention and degradation that can promote the arrest of mitochondrial motility and the restoration of mitochondrial dynamics of damaged mitochondria in neurodegenerative diseases. Summary of the Invention

[0011] The present invention provides methods and compositions for monitoring, treating, and screening for neurodegenerative conditions (e.g., Parkinson's disease) in a subject. Aspects of the methods include administering a MIRO1 reductant to a subject having Parkinson's disease. The present invention also provides a companion diagnostic assay for determining whether a subject is suitable for treatment with a MIRO1 reductant and treating the subject based on the results.

[0012] In one aspect, a method for determining the MIRO1 status of a subject is provided, which comprises measuring the response of Miro1 to mitochondrial depolarization using a biochemical assay, Western blotting, or ELISA to determine whether there is insufficient MIRO1 removal in the subject after depolarization, wherein a subject with insufficient MIRO1 removal after depolarization is selected for treatment with a MIRO1 reductant. Determining the MIRO1 status may comprise detecting the MIRO1 level in the subject and comparing the MIRO1 level with a control MIRO1 level in a control subject. In some embodiments, detecting the MIRO1 level comprises detecting MIRO1 in a tissue sample (e.g., skin fibroblasts) of the subject. In some embodiments, the method comprises the assays described in any of the examples.

[0013] The present invention provides a method for actively screening candidate agents for reducing MIRO1 levels, the method comprising: (a) contacting a candidate agent with a sample from a subject with insufficient or suspected insufficient MIRO1 removal, (b) measuring the MIRO1 level in the sample, (c) comparing the MIRO1 level in the sample with a control MIRO1 level, and (d) evaluating the activity of the candidate agent if the MIRO1 level in the sample is lower than the control MIRO1 level.

[0014] The present invention provides a method for treating a neurodegenerative disorder, which comprises administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro in sporadic Parkinson's disease cells with depolarized mitochondria by three standard deviations or more compared to the reduction of Miro in control depolarized sporadic Parkinson's disease cells not contacted with the Miro reducing agent. The healthy cells may be healthy cells with depolarized mitochondria, wherein the healthy cells are contacted with a mitochondrial depolarizing agent.

[0015] The present invention provides a method for treating a neurodegenerative disorder, which comprises administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro in Parkinson's disease cells with depolarized mitochondria by three standard deviations or more compared to the reduction of Miro in control depolarized sporadic Parkinson's disease cells not contacted with the Miro reducing agent.

[0016] The present invention provides a method for treating a neurodegenerative disorder, which comprises administering a Miro reducing agent to a subject in need thereof, wherein: (a) the Miro reducing agent reduces Miro in Parkinson's disease cells with depolarized mitochondria by two standard deviations or more compared to the reduction of Miro in control Parkinson's disease cells with depolarized mitochondria not contacted with the Miro reducing agent; and (b) the Miro 1 reducing agent reduces Miro in Parkinson's disease cells with non-depolarized mitochondria by one standard deviation or less compared to the reduction of Miro in control Parkinson's disease cells with non-depolarized mitochondria not contacted with the Miro reducing agent.

[0017] The Parkinson's disease cells may be Parkinson's disease cells with depolarized mitochondria contacted with a mitochondrial depolarizing agent. The Parkinson's disease cells may be sporadic Parkinson's disease cells. The Parkinson's disease cells may be fibroblasts.

[0018] The present invention provides a method for treating neurodegenerative disorders, which comprises administering a Miro reducing agent to a subject in need thereof, wherein in the following assay, the Miro reducing agent reduces Miro: (a) inoculating fibroblasts from a patient with sporadic Parkinson's disease into the wells of an array; (b) 24 hours after completion of step (a), adding a candidate agent to the test wells, and not adding the candidate agent to the control wells; (c) 10 hours after completion of step (b), adding FCCP to the test wells and the control wells; (d) 14 hours after completion of step (c), fixing the cells in the test wells and the control wells with ice-cold 90% methanol; and (e) immunostaining the cells in the test wells and the control wells with anti-Miro and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and imaging with a confocal microscope to measure the Miro intensity / cell for the images of the test wells and the control wells; wherein when the candidate agent reduces Miro in the test wells by three standard deviations or more relative to the control wells, the candidate agent is a Miro reducing agent.

[0019] The present invention provides a method for treating neurodegenerative disorders, which comprises administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro in sporadic Parkinson's disease cells according to the following assay: (a) inoculating fibroblasts from a sporadic Parkinson's disease patient into the wells of an array; (b) 24 hours after completion of step (a), adding a candidate agent to a first test well, and not adding the candidate agent to a first control well; (c) 10 hours after completion of step (b), adding FCCP to the first test well and the first control well; (d) 14 hours after completion of step (c), fixing the cells in the first test well and the first control well with ice-cold 90% methanol; and (e) immunostaining the cells in the first test well and the first control well with anti-Miro and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and imaging with a confocal microscope to measure the Miro intensity / cell for the images of the first test well and the first control well; and wherein the Miro reducing agent does not reduce Miro in sporadic Parkinson's disease cells according to the following assay: (a1) inoculating fibroblasts from a sporadic Parkinson's disease patient into the wells of an array; (b1) 24 hours after completion of step (a1), adding the candidate agent to a second test well, and not adding the candidate agent to a second control well; (c1) 14 hours after completion of step (b1), fixing the cells in the second test well and the second control well with ice-cold 90% methanol; and (d1) immunostaining the cells in the second test well and the second control well with anti-Miro and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and imaging with a confocal microscope to measure the Miro intensity / cell for the images of the second test well and the second control well; wherein when the candidate agent reduces Miro in the first test well by two standard deviations or more relative to the first control well, and the candidate agent reduces Miro in the second test well by less than one standard deviation relative to the second control well, the candidate agent is a Miro reducing agent.

[0020] In certain embodiments, the neurodegenerative disorder is Parkinson's disease (PD). In some such embodiments, the PD is a familial form of PD, such as PTEN-induced putative kinase 1 (PINK-1)-associated form of PD, Parkin-associated form of PD, LRRK2-associated form of PD, α-synuclein (SNCA)-associated form of PD, E3 ligase (Parkin)-associated form of PD, GBA-associated form of PD, ubiquitin carboxyl terminal hydrolase LI (UCHL1)-associated form of PD, Parkinson protein 7 (PARK7, DJ-1)-associated form of PD, ATP13A2-associated form of PD, group VI phospholipase A2 (PLA2G6)-associated form of PD. PD of the type, DnaJ (Hsp40) homolog subfamily C member 6 (DNAJC6, PARK19)-related form of PD; eukaryotic translation initiation factor 4γ-1 (EIF4G1, PARK18)-related form of PD; F-box protein 7 (FBXO7)-related form of PD; GRB10-interacting GYF protein 2 (GIGYF2)-related form of PD; HtrA serine peptidase 2 (HTRA2)-related form of PD; synaptobrevin 1 (SYNJ1)-related form of PD; and vacuolar protein sorting protein 35 homolog (VPS35)-related form of PD. In other such embodiments, the PD is a sporadic form of Parkinson's disease, for example, it is associated with a sporadic mutation in one of the above genes. In certain such embodiments, the MIRO1 reducing agent is administered to the midbrain and / or putamen of the subject.

[0021] In some embodiments, an assay for determining the MIRO1 status of a subject is provided. In some embodiments, a patient cell population (for convenience, fibroblasts are used) is assayed to identify the MIRO1 phenotype of the subject. Miro1 is located on the surface of the mitochondria and mediates mitochondrial movement. Miro1 in depolarized mitochondria is removed to promote its clearance (clearance by mitochondrial autophagy). Measuring the response of Miro1 to mitochondrial depolarization using biochemical assays, ELISA, etc. shows that a considerable proportion of Parkinson's disease subjects have insufficient MIRO1 removal after depolarization.

[0022] As described in the examples herein, MIRO1 status is associated with the presence of certain neurodegenerative disorders. For example, elevated MIRO1 levels are associated with Parkinson's disease.

[0023] The Miro reducing agent can reduce the intracellular calcium level by 20% or more. The molecular weight of the Miro reducing agent can be from 100 to 2000 daltons. The Miro reducing agent can be an antibody, a peptide, a protein, or a part of one of them. The Miro reducing agent can bind to an EF hand protein. The Miro reducing agent can be a calcium channel blocker. The calcium channel blocker can be an L-type and an N-type calcium channel blocker. The subject can have an elevated Miro. The subject can otherwise have no symptoms of a neurodegenerative disorder. The neurodegenerative disorder can be Parkinson's disease.

[0024] The present invention provides a method for selecting a subject to receive a therapeutic agent for treating a neurodegenerative disorder, comprising: (a) collecting cells from the subject and evaluating a first control portion of the cells for the pre-depolarization Miro level in the cells; (b) contacting a second test portion of the cells with a depolarizing agent; and (c) evaluating the post-depolarization Miro level in the second test portion of the cells contacted with the depolarizing agent and comparing the Miro level with the pre-depolarization Miro level in the first control portion of the cells; wherein when the post-depolarization Miro level in the second test portion of the cells is reduced by 40% or less relative to the pre-depolarization Miro level in the first control portion of the cells, treating the subject's neurodegenerative disorder with a therapeutic agent. The present invention provides a method for selecting a subject to receive a therapeutic agent for treating a neurodegenerative disorder, wherein when the post-depolarization Miro level in the second test portion of the cells is reduced by 10% to 50% relative to the pre-depolarization Miro level in the first control portion of the cells, treating the subject's neurodegenerative disorder with a therapeutic agent.

[0025] The neurodegenerative disorder can be Parkinson's disease. The subject can have no symptoms of Parkinson's disease. The therapeutic agent can be selected from levodopa and dopamine antagonists. The therapeutic agent can be a Miro reducing agent. The molecular weight of the therapeutic agent can be from 100 to 2000 daltons. The therapeutic agent can be an antibody, a peptide, a protein, or a part of one of them. The therapeutic agent can bind to an EF hand protein. The therapeutic agent can be a calcium channel blocker. The calcium channel blocker can be an L-type and an N-type calcium channel blocker.

[0026] The present invention provides a method for improving age-related conditions, comprising administering a Miro reducing agent to a subject in need thereof. The age-related conditions can be selected from memory impairment, muscle degeneration, arthritis, cardiovascular disease, osteoporosis, glaucoma, dementia, macular degeneration, and cataracts. The Miro reducing agent can be administered prophylactically.

[0027] Incorporated by reference

[0028] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Before describing the methods and compositions of the present invention, it should be understood that aspects of the present invention are not limited to the specific methods or compositions described, as there will surely be differences in actual implementation. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to limit the inventive concept, and the scope of the present invention will be defined only by the appended claims.

[0030] The novel features of the present invention are set forth in detail in the appended claims. The features and advantages of the present invention can be better understood by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the present invention are utilized, as well as the accompanying drawings, in which:

[0031] Figure 1A-1F . Response of Miro1 to CCCP in fibroblasts. Figure 1A : Illustration of our readings. Figure 1B-1C : Examples of readings obtained using healthy-1, PD-2, and risk-2. Figure 1D : Heat map showing relative mitochondrial protein levels. The intensity of each band in the mitochondrial fraction was normalized based on the intensity of the mitochondrial loading control VDAC from the same blot and expressed as a ratio to the mean of Healthy-1 treated with DMSO; this control was included in each experiment. The mean values were imported into the heat map. n = 3 - 35. Figure 1E-1F : ELISA for Miro1. n = 4 (including duplicates) / run. Comparison within the same subject. Whole study

[0032] *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0033] Figure 2 A-2H shows the relationship between the Miro1 ratio (CCCP / DMSO) and different variables. Figure 2 A: Healthy subjects vs. Parkinson's disease subjects ("PD"); Figure 2 B: Male subjects vs. female subjects; Figure 2 C: Sampling age (years); Figure 2 D: Age of onset of PD (years); Figure 2 E: PD progression (number of years with PD); Figure 2 F: UPDRS; Figure 2 G: Hoehn and Yahr scale for PD subjectsFigure 2 H: Mini-Mental State Examination of PD subjects.

[0034] Figure 3 A-3G: Figure 3 A shows the relative β-actin level or Miro1 level (ng / ml) in the subjects; Figure 3 B shows the relationship between the absorbance at 450 nm and the β-actin level; Figure 3 C shows the relationship between the relative β-actin level and the capture antibody; Figure 3 D shows the relationship between the absorbance at 450 nm and the Miro1 level; Figure 3 E shows the relationship between the relative Miro1 level and the capture antibody; Figure 3 F shows the relationship between the absorbance at 450 nm and Miro1 (ng / ml); Figure 3 F shows the relationship between Miro1 (ng / ml) and the capture antibody. DETAILED DESCRIPTION

[0035] In one aspect, the present invention provides a method for diagnosing a neurodegenerative disorder (e.g., Parkinson's disease), which comprises detecting Miro, e.g., Miro1, in a subject. In certain embodiments, the level of Miro1 in the subject, e.g., the level of Miro1 in a tissue sample from the subject, is detected using the assays described herein. The level of Miro1 in the subject can be compared with the control Miro1 level in a control subject to determine whether a neurodegenerative disorder is present. For example, if the Miro1 level is higher (e.g., 20% or more or 30% or more) than the control Miro1 level, it may indicate the presence of Parkinson's disease. If a neurodegenerative disorder is indicated, the neurodegenerative disorder of the subject can be treated, e.g., with a treatment regimen known in the art or described herein.

[0036] In one aspect, the present invention provides a method for treating a neurodegenerative disorder (e.g., Parkinson's disease), which comprises administering a Miro reductant, e.g., a Miro1 reductant, to a subject in need thereof. In certain embodiments, the Miro reductant is identified using the assays described herein. In certain embodiments, the Miro reductant reduces the level of Miro (e.g., Miro1) in a cell, e.g., a cell from a subject in whom Miro1 removal is insufficient or suspected to be insufficient. In certain embodiments, the Miro reductant reduces the level of Miro (e.g., Miro1) in a cell to a level consistent with the level of Miro (e.g., Miro1) in a control cell.

[0037] In certain embodiments, the present invention provides methods for treating a subject having a neurodegenerative disorder (e.g., Parkinson's disease), wherein the subject is diagnosed with Parkinson's disease using the methods described herein. Methods for treating neurodegenerative disorders with Miro reductants are described below.

[0038] In certain embodiments, the present invention provides methods for identifying Miro1 reductants. In certain embodiments, the present invention provides methods for screening candidate agents for treating neurodegenerative disorders (e.g., Parkinson's disease) using the assays described herein.

[0039] Before further describing the present invention, it is to be understood that the invention is not limited to the particular embodiments described, as variations will undoubtedly occur in actual practice. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the inventive concept, the scope of the invention being defined solely by the appended claims.

[0040] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of that range, as well as any other stated value or intermediate value within that range, is encompassed within the invention. Unless the context clearly dictates otherwise, each intermediate value is to be taken as low as one-tenth of the unit of the lower limit. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limits within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the invention.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the publications cited.

[0042] As used herein, terms such as "treatment" generally refer to obtaining a desired pharmacological and / or physiological effect. Preventive effects refer to the complete or partial prevention of a disease or its symptoms, and therapeutic effects refer to the partial or complete cure of a disease and / or the adverse reactions caused by the disease. "Treatment" as used herein encompasses any treatment of a mammalian disease, including: (a) preventing a subject from developing a disease, where the subject may be susceptible to the disease but has not been diagnosed; (b) inhibiting the disease, i.e., preventing its progression; or (c) alleviating the disease, i.e., causing the disease to regress. The therapeutic agent can be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of current diseases, where the treatment can stabilize or alleviate the adverse clinical symptoms of the subject. Ideally, such treatment is carried out before the complete loss of function of the affected tissue. Ideally, the subject therapy will be implemented during the symptomatic phase of the disease (and in some cases, after the symptomatic phase of the disease).

[0043] The terms "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject in need of diagnosis, treatment, or therapy, particularly a human being.

[0044] It must be noted that, as used in this patent and the appended claims, the singular forms of "a", "an", and "the" are to be construed as including the plural unless the context clearly dictates otherwise. It should also be noted that claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as a basis for the use of special terms such as "solely", "only", etc. in relation to the recitation of claim elements or the use of "negative" limitations.

[0045] The terms "candidate agent", "test agent", "agent", "substance", and "compound" are used interchangeably herein. Candidate agents encompass a wide variety of chemical classes and are typically synthetic, semi-synthetic, or naturally occurring inorganic or organic molecules. Candidate agents include agents found in large synthetic or natural compound libraries.

[0046] Mitochondrial Rho (Miro) is a small GTPase, well-known for its role in mitochondrial transport and homeostasis. Miro is a highly conserved protein in single-celled and multicellular eukaryotes. Miro1 and Miro2 are conserved single-pass transmembrane integral proteins with their N-terminal regions exposed to the cytoplasmic side, consisting of two GTPase domains separated by a pair of canonical EF hands. Miro also contains a C-terminal hydrophobic domain that permits membrane insertion. The Miro GTPase is localized on the outer mitochondrial membrane and plays a key role in intracellular mitochondrial movement in metazoans, especially in long-distance movement along microtubule bundles in neurons. Miro is also known to play a role in intercellular mitochondrial transport between cells via tunneling nanotubes. Miro mediates bidirectional movement of mitochondria along microtubule bundles by engaging with kinesin and dynein. Miro engages with microtubule bundles via cargo adaptors of the Milton / Trak family. Miro also uses the mitochondrial actin motor Myo19 to transport mitochondria along actin tracks. Miro is also involved in peroxisome distribution by binding to Peroxin 26. In addition, Miro plays a role in mitochondrial fusion and fission dynamics by regulating the mitochondrial dynamin Drp1 and interacting with the mitochondrial fusion proteins mitofusins 1 and 2.

[0047] Miro turnover is regulated by the PTEN-induced putative kinase 1 (PINK1) / Parkin pathway. PINK1 phosphorylates Miro, thereby promoting its interaction with the E3 ubiquitin ligase Parkin. Parkin promotes Miro ubiquitination and degradation, thus effectively preventing axonal transport of damaged mitochondria. On the other hand, PINK1-phosphorylated Miro also recruits Parkin to the damaged mitochondria, which are then marked for mitophagic destruction.

[0048] Miro is located in the outer mitochondrial membrane and anchors the microtubule motors kinesin and dynein to mitochondria. After mitochondrial damage caused by mitochondrial depolarization occurs, the cell initiates mitophagy. Mitochondrial movement stops before the onset of mitophagy, which is a cellular mechanism by which depolarized mitochondria are degraded by autophagosomes and lysosomes. The movement arrest can sequester damaged mitochondria, prevent the movement of damaged mitochondria, and at the same time prevent the reintroduction of damage to other healthy mitochondria. Removal of Miro from the surface of damaged mitochondria, followed by its degradation by the proteasome, enables this depolarization-triggered mitochondrial arrest. However, in skin fibroblasts from subjects with familial and sporadic PD, significant defects were observed in the degradation of Miro and subsequent clearance of damaged mitochondria.

[0049] The EF hands of Miro mediate Ca 2+Dependent bidirectional mitochondrial transport arrest. Miro ligates with KIF5 to mediate mitochondrial transport. 2+ The binding causes KIF5 to dissociate from the mitochondria, or "sever" the connection between KIF5 and the microtubules. 2+ Binding to the EF-hand triggers a direct interaction of the motor domain with Miro, thereby preventing the motor from engaging MTs.

[0050] As used herein, "Miro" may refer to any or all of the following: family members, isoforms, homologs, paralogs, mutants, alleles, variants, derivatives, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide chains, etc. Miro refers to the human sequence Miro, such as the complete amino acid sequence of a human Miro isoform having the Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7. The human Miro sequence is different from the human Miro isoform with Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6 or Q8IXI2-7 in that, for example, it has a conservative mutation or a mutation in a non-conservative region, and Miro has substantially the same biological function as the human Miro isoform with identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6 or Q8IXI2-7. Miro is also known as used RHOT1 and mitochondrial Rho GTPase1. Miro includes isomers of Miro, such as Miro1 and Miro2.

[0051] The specific Miro sequence can be at least 90%, at least 95%, or even at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of Miro with Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7. In certain embodiments, the human Miro sequence may differ from the Miro sequence with Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7 by more than 10 amino acid differences. In certain embodiments, the human Miro may show no more than 5, or even no more than 4, no more than 3, no more than 2, or no more than 1 amino acid difference compared to the Miro sequence with the Uniprot identifier Q8IXI2-1, Q8IXI2-2, Q8IXI2-3, Q8IXI2-4, Q8IXI2-5, Q8IXI2-6, or Q8IXI2-7. The percent identity may be determined as described herein.

[0052] An "isoform" of a protein can be, for example, a protein formed by alternative splicing of a gene expressing the protein, or a degradation product of the protein. "Sequence homology" refers to a nucleotide-to-nucleotide correspondence between two polynucleotides or an amino acid-to-amino acid correspondence between two polypeptide sequences. For two nucleic acid sequences or amino acid sequences, "sequence identity" or "identity" refers to the residues in the two sequences that are the same when aligned for maximum consistency over a specified comparison window.

[0053] As used herein, "sequence identity percentage" refers to a value determined by comparing two optimally aligned sequences within a comparison window, wherein the polynucleotide or polypeptide sequence portion within the comparison window may contain additions or deletions, i.e., gaps, compared to a reference window that does not contain additions or deletions for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions of identical nucleotides or amino acids present in both sequences to obtain the number of matching positions; dividing the number of matching positions by the total number of positions given within the comparison window; and then multiplying the result by 100 to obtain the sequence identity percentage.

[0054] Sequence comparison (e.g., to assess identity) can be performed by any suitable alignment algorithm, including but not limited to Needleman-Wunsch algorithm (see, e.g., EMBOSS Needle aligner, available from www.ebi.ac.uk / Tools / psa / emboss_needle / , optionally with default settings), BLAST algorithm (see, e.g., BLAST alignment tool, available from blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings) and Smith-Waterman algorithm (see, e.g., EMBOSS Water aligner, available from www.ebi.ac.uk / Tools / psa / emboss_water / , optionally with default settings). Optimal alignment can be assessed using any suitable parameters (including default parameters) of the selected algorithm.

[0055] The "percent identity" between two sequences can be calculated as follows: the number of exact matches between the two optimally aligned sequences is divided by the length of the reference sequence and then multiplied by 100. Percent identity can also be determined by, for example, comparing sequence information using an advanced BLAST computer program (including version 2.2.9, available from the National Institutes of Health). The BLAST program can be based on the alignment method in the following publications: Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990); Altschul et al., J. Mol. Biol., 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). Briefly, the BLAST program may define identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine the percent identity of the sequences being compared over the entire length. Default parameters may be provided to optimize searches of short query sequences, such as in the case of using the blastp program. The program may also allow the use of a SEG filter to mask segments of the query sequence, as determined by the SEG program in the following publication: Wootton and Federhen, Computers and Chemistry, 17: 149-163 (1993). High sequence identity may include sequence identities ranging from about 80% to 99% and integer values ​​between the ranges.

[0056] "Homologue" may refer to any sequence having a sequence homology of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% to another sequence. In certain embodiments, a homologue has a sequence homology of 70% to 99%, 80% to 99%, 85% to 99%, 90% to 99%, or even 95% to 99% to another sequence. In some cases, the homologue may have equivalence with an original or naturally occurring sequence in function or structure. In some cases, the homologue may have equivalence with a domain, a motif or a portion of the protein encoded by an original or naturally occurring sequence in function or structure.

[0057] A sequence comparison program can be used to perform homology comparison. A computer program can calculate the homology percentage (%) between two or more sequences, or it can calculate the common sequence identity of two or more amino acid or nucleic acid sequences. Sequence homology can be generated by any of many computer programs, such as BLAST or FASTA, etc. A suitable computer program for performing such comparisons is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research, 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999, ibid., Chapter 18), FASTA (Atschul et al., 1990, Journal of Molecular Biology, 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al., 1999, ibid., pages 7-58 to 7-60).

[0058] The percent homology can be calculated for consecutive sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "ungapped" alignment. Typically, such ungapped alignments can be performed over a relatively small number of residues.

[0059] In other identical sequence pairs, an insertion or deletion may cause the subsequent amino acids or nucleotide residues to no longer align, which may result in a substantial reduction in the homology percentage when performing a global comparison. Therefore, the sequence comparison method can be designed to produce an optimal comparison that takes into account possible insertions and deletions without excessively penalizing the overall homology or identity score. This can be achieved by inserting "gaps" in the sequence comparison in an attempt to maximize local homology or identity.

[0060] Calculation of the maximum homology percentage can utilize optimal alignment taking into account gap penalties. Another tool, BLAST 2 Sequences, can also be used to compare protein and nucleotide sequences (see FEMS Microbiology Letters, 1999 174(2):247-50; FEMS Microbiology Letters, 1999 177(1):187-8; and the National Center for Biotechnology Information website on the National Institutes of Health website).

[0061] Homologous sequences may also have deletions, insertions or substitutions of amino acid residues, thereby producing functionally equivalent substances. Intentional amino acid substitutions may be made based on the similarity of amino acid properties (e.g., polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues), and therefore, it is useful to group amino acids together in functional groups. Amino acids may be grouped together based solely on the properties of their side chains. A group of conservative amino acids may be described in the form of a Venn diagram (Livingstone C.D. and Barton GJ, (1993) "Protein sequence alignment: a hierarchical analysis strategy for residue conservation", Computer Applications in Biological Sciences, 9: 745-756) (Taylor WR, (1986) "Classification of amino acid conservation", Journal of Theoretical Biology, 119; 205-218).

[0062] "Miro reducing agent" (e.g., MIRO1 reducing agent) refers to any agent that reduces the level of Miro protein or its homologues in cells with depolarized mitochondria. In an exemplary embodiment, the Miro reducing agent can reduce at least one biological activity of Miro protein in cells with depolarized mitochondria. Exemplary biological activities of Miro include promoting mitochondrial transport, mitochondrial autophagy, microtubule binding, mitochondrial fission and fusion, etc. The Miro reducing agent can be, for example, a small molecule, a peptide, an aptamer, a protein, or a functional fragment of a protein. The functional fragment of a protein used herein refers to all or part of the molecular elements in a protein that affect a specific function (e.g., protein binding, signal transduction, etc.).

[0063] In some embodiments, a MIRO1 reducing agent inhibits the level or biological activity of MIRO1 by 20% or more, such as 30% or more, 40% or more, or 50% or more, and sometimes by 60% or more, 70% or more, or 80% or more, such as 90%, 95%, or 100%, relative to an untreated control that has not been contacted with the reducing agent. The reducing agent can be validated by any suitable method in the art for detecting the level and / or activity of MIRO1 in the presence and absence of a MIRO1 reducing agent.

[0064] For example, the level and / or phosphorylation state of the Miro protein (Ser156, Thr298 or Thr299 of Miro1 and Miro2, see, e.g., Wang et al., Cell, 2011, 147(4): 893-906) can be detected by, for example, immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a phospho-specific or universal antibody, wherein increased phosphorylation of the Miro protein and / or decreased levels of total Miro protein or decreased Khc phosphorylation after contact with the agent can indicate that the agent can treat Parkinson's disease. As another example, the level and / or ubiquitination of the Miro protein can be detected by, for example, immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a ubiquitin-specific antibody, wherein increased ubiquitination after contact with the candidate agent indicates that the agent can treat Parkinson's disease. As another example, the ability of the target mitochondrial protein to transport mitochondria within a cell can be assessed by, for example, treating cultured cells (e.g., neurons) with the MIRO1 reducing agent and observing the transport of mitochondria in the cells compared to cells not treated with the MIRO1 reducing agent using techniques such as live cell imaging (see, for example, Brickley and Stephenson, Journal of Biological Chemistry, 286(20):18079-92 (2011); Misko et al., Journal of Neuroscience, 30(19):4232-40 (2010); Russo GJ et al., Journal of Neuroscience, 29(17):5443-55 (2009)). As another example, since the formation of a complex between Miro (e.g., Miro 1 and 2), TRAK (e.g., TRAK1 and 2), and Khc is essential for mitochondrial transport in neurons (see, e.g., Brickley and Stephenson, Journal of Biological Chemistry, 286(20):18079-92 (2011)), the effect of a MIRO1 reducing agent on Miro function can be assessed by assessing the ability of Miro, TRAK, and Khc to form a complex in the presence of a MIRO1 reducing agent. Such an assessment can be performed using any technique to determine protein-protein interactions, including, but not limited to, co-immunoprecipitation and affinity purification techniques. In a specific embodiment, the ability is assessed in cells with a familial PD mutation (e.g., a PINK1 or LRRK2 mutation).

[0065] "Mitochondrial depolarization" is the process by which the mitochondrial membrane potential changes from the resting potential to the depolarized direction, from negative to positive. Normal, mild influx of calcium from the cytosol into the mitochondrial matrix results in a transient depolarization that can be corrected by pumping out protons. Chemically induced mitochondrial depolarization provides a suitable assay for determining the effects of mitochondrial damage.

[0066] Many agents are known agents that can be used experimentally to cause mitochondrial depolarization and can be used in the methods of the invention for such purposes. Such agents generally fall into the category of mitochondrial uncouplers or uncoupling agents, which disrupt oxidative phosphorylation in mitochondria by dissociating ATP synthesis reactants from the electron transport chain. As a result, mitochondria consume energy to generate proton motive force, but the proton motive force is dissipated before the ATP synthase can recapture the energy and use it to make ATP.

[0067] The concentration and exposure time of the agent are sufficient to uncouple or depolarize the mitochondria. For example, a concentration of about 10-100 μM (e.g., about 20 to 80 μM, about 30 to 50 μM) of CCCP is sufficient. The dosage of other agents can provide activity comparable to these CCCP concentrations. The cells are incubated for a sufficiently long time to depolarize the mitochondria and begin to clear, for example, at least about 1 hour, at least about 2 hours, usually no more than about 24 hours, and can be about 1 to 24 hours, about 2 to 20 hours, about 3 to 18 hours, about 4 to 14 hours, about 5 to 10 hours, and can be about 4 to 8 hours.

[0068] Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide-3-chlorophenylhydrazone (CCCP) are commonly used mitochondrial uncouplers in research. These molecules are lipophilic weak acids that act as proton carriers. Due to their hydrophobic nature, these compounds can easily cross biological membranes and allow the protons to pass through these membranes. Other molecules suitable for this purpose include 1,3-bis(3,5-dichlorophenyl)urea; dodecyltriphenylphosphonium; dinitrophenol; BAM15 ((2-fluorophenyl)6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)amine) - a mitochondrial-specific proton carrier uncoupler with potency similar to FCCP or DNP; FR58P1 (bromoalkyl ester of a hydroquinone derivative) - another mitochondrial proton carrier.

[0069] Other mitochondrial-specific uncouplers include MitoFluo, which is a conjugate of a triphenylphosphonium cation and fluorescein, acting as a fluorescent uncoupler that preferentially accumulates in mitochondria; dodecyltriphenylphosphonium (C12TPP), which acts as a fatty acid anion carrier, promoting fatty acid recycling across the membrane, thereby achieving mitochondrial uncoupling; rhodamine 19-butyl ester C4R1, which acts as a mild mitochondrial uncoupler; and MitoPhotoDNP, which is a fusion of DNP, an o-nitrobenzyl group (a photoactivatable group), and triphenylphosphonium. Bupivacaine (a local anesthetic) can partially act as a proton carrier, but can also inhibit state 3 respiration by altering the stoichiometry of the mitochondrial proton pump. Weak CH acids, such as o-carborane (1,2-C 2 B 10 H 12 ), also has global uncoupling properties when used at concentrations comparable to FCCP (10 μM range).

[0070] Affinity assays (usually immunoassays) are assays or analytical procedures that rely on the binding of a target molecule (i.e., Miro1) to a receptor, antibody, or other macromolecule. Detection methods are used to determine the presence and amount of the binding complex formed. Many forms of such assays are known and used in the art and are suitable for detecting Miro1 degradation after mitochondrial uncoupling or depolarization. In some embodiments, the assay format is suitable for high-throughput analysis.

[0071] Suitable assay formats include immunoassays using Miro1-specific antibodies. Suitable antibodies for this purpose are known and commercially available as polyclonal or monoclonal compositions, such as antibodies from Invitrogen, including monoclonal CL1095, CL1083; antibodies from Sigma Aldrich, including clone 4H4, Santa Cruz Biotechnology anti-Rho T1 antibody (A-8); and the like.

[0072] The assay of interest includes, for example, Western blotting; immunohistochemistry; immunoprecipitation; and the like, and particularly includes immunoassays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA); enzyme immunoassay (EIA).

[0073] Enzyme-linked immunosorbent assays (ELISAs) are used to qualitatively and quantitatively analyze the presence or concentration of a specific soluble antigen (e.g., Miro1) in a liquid sample (e.g., cell lysate). These assays typically utilize the ability of a multiwell plate or other plate to bind to an antibody that captures the cognate antigen. A colorimetric endpoint that can be detected by an absorbance wavelength and quantified based on a known antigen or antibody dilution standard curve is typically used. The detection antibody is typically labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase, a fluorescent marker, an electrochemiluminescent marker, or through an intermediate marker such as biotin.

[0074] Common ELISA formats include sandwich ELISAs, so named because the analyte is "sandwiched" between two different antibodies. The capture substrate in this format is a capture antibody, usually a monoclonal antibody, to increase the specificity of the assay and reduce background noise. The analyte binds to the capture antibody and is then detected by binding to a detection antibody. A variant of the sandwich ELISA assay, called a single molecule assay (Simoa), uses magnetic beads coated with capture antibodies; each bead binds to one or zero target molecules, and individual beads are detected with another antibody (the detection antibody) and a labeled enzyme.

[0075] Other ELISA formats include indirect ELISA, in which the capture substrate is the specific antigen being tested, and the detection step is mediated by a primary antibody and an enzyme-conjugated secondary antibody that reacts with the primary antibody. Thus, the primary antibody that recognizes the antigen is not labeled. In direct ELISA, the capture substrate is the specific antigen being tested, and the enzyme that catalyzes the color change reaction is conjugated to the antigen-detecting antibody.

[0076] Immuno-PCR (I-PCR) is a technique that combines the sensitivity of nucleic acid amplification (PCR assay) with the specificity of antibody-based assays, thereby increasing detection sensitivity.

[0077] Screening method

[0078] In some aspects of the invention, methods are provided for screening candidate agents for activity in treating Parkinson's disease in subjects with Parkinson's disease. To this end, it has been demonstrated that mitochondrial transporters (e.g., Miro proteins, trafficking kinases, and kinesin heavy chains) promote the development or progression of Parkinson's disease or its symptoms. Therefore, screening candidate agents that inhibit the expression or activity of mitochondrial transporters in cells should identify agents that can be used to treat Parkinson's disease in subjects. In detail, the ability of the agent to reduce Miro1 levels after mitochondrial depolarization, and preferably without significantly reducing basal Miro1 levels, is tested.

[0079] The test agents may be obtained using any of a number of combinatorial library approaches known in the art, including: biological libraries; spatially addressable parallel solid or solution phase libraries; synthetic library approaches requiring deconvolution; "one bead one compound" library approaches; and synthetic library approaches using affinity chromatography selection.

[0080] In one embodiment, an agent that changes Miro1 degradation is identified; a cell (e.g., a living cell or a cell population) is contacted with the test agent; alternatively, the protein can be directly contacted with the test agent. The activity level (amount) is assessed directly or indirectly and compared to the activity level in a control (e.g., in the absence of the test agent). If the amount of difference between the activity level in the presence of the agent and the activity level in the absence of the agent is statistically significant, the agent is an agent that changes the activity.

[0081] The present invention also relates to novel agents identified by the above-described screening assays. Therefore, it is also within the scope of the present invention to further use the agents identified as described herein in the treatment methods described herein. For example, the agents identified as described herein can be used to alter Miro1 degradation by the agents identified as described herein.

[0082] For example, in a screening assay for bioactive agents, cells expressing a mitochondrial transporter of interest are contacted with a candidate agent of interest, and the effect of the candidate agent on the expression or function of the mitochondrial transporter is assessed by monitoring one or more mitochondrial-related parameters. Specifically, the activity of the candidate agent can be assessed by determining the level of Miro1 after mitochondrial depolarization.

[0083] Parameters are quantifiable components of cells, especially components that can be accurately measured in high-throughput systems. Parameters can be any cellular component or cell product, including cell surface determinants, receptors, proteins or their conformations or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids (e.g., mRNA, DNA, etc.) or parts or combinations thereof derived from such cellular components. Although most parameters provide quantitative readings, in some cases, semi-quantitative or qualitative results are acceptable. Readings can include a single determined value, or can include an average, median, or variance, etc. Characteristically, a series of parameter readings can be obtained for each parameter derived from multiple identical determinations. The corresponding values ​​are expected to change, and standard statistical methods and common statistical methods for providing a single value are used to obtain the value range of each parameter in the test parameter group. Therefore, for example, a method of this type may include contacting a cell expressing a mitochondrial transporter with a candidate agent; and comparing the mitochondrial-related parameters with mitochondrial-related parameters in cells expressing the mitochondrial transporter but not contacted with the candidate agent, wherein the parameter difference in the cell contacted with the candidate agent indicates that the candidate agent can treat Parkinson's disease.

[0084] An example of a mitochondrial-related parameter that can be quantified when screening for agents that can treat Parkinson's disease is the phosphorylation state of Miro protein (Ser156, Thr298 or Thr299 of Miro1 and Miro2), TRAK protein or Khc (Ser residue, see, e.g., Lee and Hollenbeck, Journal of Biological Chemistry, 1995, 270(10):5600-5): for example, by immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a phospho-specific or universal antibody, wherein an increase in Miro protein phosphorylation and / or a decrease in total Miro protein levels or a decrease in Khc phosphorylation after contact with the candidate agent indicates that the candidate agent can treat Parkinson's disease. Another example of a parameter is the ubiquitination state of Miro protein, TRAK protein or Khc determined by, for example, immunoprecipitation with a mitochondrial transport protein-specific antibody followed by Western blotting with a ubiquitin-specific antibody, wherein ubiquitination increases after contact with the candidate agent, indicating that the agent can treat Parkinson's disease. Another example is the rate at which mitochondria are transported around the cell, which can be measured by, for example, live cell imaging, wherein the transport rate decreases after the cell is contacted with a candidate agent, indicating that the agent can treat Parkinson's disease. Another example is the length of the mitochondria in the cell, wherein the length of the mitochondria is shortened after the cell is contacted with a candidate agent, indicating that the agent can treat Parkinson's disease. Other output parameters may include parameters reflecting the ability of Miro protein, TRAK protein and KHC to form a complex in the presence of a MIRO1 reducing agent (see, e.g., Brickley and Stephenson, Journal of Biological Chemistry, 286(20): 18079-92 (2011)), which can be assessed by techniques such as co-immunoprecipitation or affinity purification, wherein a reduction in complex formation after contacting the cells with a candidate agent indicates that the agent can treat Parkinson's disease. In some cases, one parameter is measured. In some cases, multiple parameters are measured.

[0085] All cells contain mitochondria, therefore, any cell can be used in the target screening method. In some cases, the cell is a cell type commonly affected by Parkinson's disease, such as a muscle cell or a neuron, such as a motor neuron. In some cases, the cell contains a genetic mutation associated with Parkinson's disease, i.e., a mutation in a nuclear or mitochondrial gene. In some cases, the cell from a subject with Parkinson's disease can be acutely cultured.

[0086] Purpose candidate agents are bioactive agents covering many chemical categories, mainly organic molecules, which can include organometallic molecules, inorganic molecules, gene sequences, etc. An important aspect of the present invention is to evaluate candidate drugs, select therapeutic antibodies with preferred biological response functions and protein-based treatments. Candidate agents include functional groups necessary for structural interactions (especially hydrogen bonding) with proteins, and generally include at least one amino, carbonyl, hydroxyl or carboxyl group, often including at least two functional chemical groups. The candidate agents generally include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted by one or more of the above-mentioned functional groups. Candidate agents can also be derived from biomolecules including peptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0087] It includes pharmacologically active drugs, genetically active molecules, etc. The target compound includes a chemotherapeutic agent, an anti-inflammatory agent, a hormone or a hormone antagonist, an ion channel modifier, and a neuroactive agent. Examples of pharmaceutical preparations suitable for the present invention are those described in the following publications: "Pharmacological Basis of Therapeutics", Goodman and Gilman, McGraw-Hill, New York, New York, (1996), Ninth Edition, the following chapters: Drugs Acting on Synapses and Neuroeffector Junctions; Drugs Acting on the Central Nervous System; Self-effective substances: Drug treatment of inflammation; Water, salts and ions; Drugs affecting renal function and electrolyte metabolism; Cardiovascular drugs; Drugs affecting gastrointestinal function; Drugs affecting uterine motility; Chemotherapy for parasitic infections; Chemotherapy for microbial diseases; Chemotherapy for neoplastic diseases; Drugs for immunosuppression; Drugs acting on hematopoietic organs; Hormones and hormone antagonists; Vitamins, Dermatology; and Toxicology, all of which are incorporated herein by reference.

[0088] Test compounds include all types of molecules mentioned above, and may further include samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources (e.g., plants). Although many samples contain compound solutions, solid samples soluble in suitable solvents can also be measured. Target samples include environmental samples, such as groundwater, seawater, mining waste, etc.; biological samples, such as lysates prepared from crops, tissue samples, etc.; manufacturing samples, such as time courses during drug preparation; and compound libraries prepared for analysis; etc. Target samples include compounds that are being evaluated for potential therapeutic value, i.e., candidate drugs.

[0089] Antibodies and small molecules can be screened using a variety of methods to detect changes in MIRO1 levels in vitro and in vivo, for example, ELISA assays, etc. These methods include, but are not limited to, methods for measuring binding affinity to a target, the biodistribution of the compound in an animal or cell, or compound-mediated cytotoxicity. As a first test, an antibody or small molecule can be tested to establish interaction with a target that indicates a change in MIRO1 levels. After selective binding is established, the appropriate activity of the candidate antibody can be tested in an in vivo model. These and other screening methods known in the art provide information about the ability of a compound to bind to, modulate, or otherwise interact with a specified target, and the method is a measure of the efficacy of the compound. In some embodiments, the screening method comprises an assay described in any one of the examples.

[0090] Diagnostic method

[0091] In many aspects, the subject is screened to determine whether the subject suffers from or is susceptible to a neurodegenerative disorder (e.g., Parkinson's disease). The screening method comprises behavioral, biophysical, biochemical and imaging measurements and observations and questionnaires to determine whether the subject is at risk of developing an early neurodegenerative disorder (e.g., Parkinson's disease) or suffers from the early disease. Biophysical and behavioral observations can be evaluated independently, such as a physical examination of the subject for the external symptoms of the disease, or the evaluation can be performed in combination with questionnaires and biochemical / imaging measurements. Each individual measurement can also be used alone, or in combination with biophysical evaluations or other tests known in the art and associated with a specific neurodegenerative disorder / disorder. Examples of biochemical measurements include genetic screening for mutations and / or polymorphisms (e.g., SNP analysis, short tandem repeat analysis), biomarker-based measurements, protein expression measurements, immunohistochemical measurements, or any combination thereof. Materials for biochemical measurements can be sampled from all body fluids and tissues. Common body fluids include, but are not limited to, blood, serum, plasma, saliva, urine, gastric and digestive juices, tears, feces, semen, vaginal fluid, interstitial fluid from tumor tissue, and cerebrospinal fluid. Methods for obtaining body tissue and body fluid samples include, but are not limited to, biopsy, cheek swab, nasal swab, rectal swab, skin fat extraction, or other collection strategies for obtaining biological or chemical substances. In some embodiments, the sample is a tissue sample. For example, the tissue sample can be fibroblasts, such as skin fibroblasts.

[0092] A control value is a measurement of a corresponding control sample from a control (ie, non-diseased) subject. For example, in some embodiments, MIRO1 levels in skin fibroblasts from a subject are compared to control MIRO1 levels in control skin fibroblasts from a control subject.

[0093] Antibodies and small molecule inhibitors can be screened using a variety of methods to detect target binding in vitro and in vivo. For example, ELISA assays, etc. These methods include, but are not limited to, methods for measuring binding affinity to the target, the biodistribution of the compound in animals or cells, or compound-mediated cytotoxicity. As a first test, the binding of the antibody or small molecule to the target mitochondrial transporter can be tested. After selective binding is established, the appropriate activity of the candidate antibody can be tested in an in vivo model. These and other screening methods known in the art provide information about the ability of the compound to bind, modulate, or otherwise interact with a specified target, and the method is a measure of the efficacy of the compound.

[0094] (Subject subject subject) The method for determining the MIRO1 status of a subject may include measuring the response of the Miro1 to mitochondrial depolarization using a biochemical assay, Western blot, or ELISA to determine whether the subject has insufficient MIRO1 removal after depolarization, wherein subjects with insufficient MIRO1 removal after depolarization are selected to receive treatment with a MIRO1 reducing agent. Determining the MIRO1 status may include detecting the level of MIRO1 in a subject, and comparing the MIRO1 level with a control MIRO1 level in a control subject. In some embodiments, detecting the level of MIRO1 includes detecting MIRO1 in a tissue sample (e.g., skin fibroblasts) of the subject. In some embodiments, the method includes the assay described in any one of the examples.

[0095] The method may further comprise treating the subject with the MIRO1 reducing agent. In such cases, the MIRO1 level may be monitored before and / or after treatment with the MIRO1 reducing agent. In some embodiments, the MIRO1 reducing agent is administered to the midbrain and / or putamen of the subject. The treatment may comprise administering to the subject a therapeutically effective amount of one or more agents selected from levodopa, dopamine agonists, MAO-B inhibitors, amantadine or anticholinergics before, during or after administration of a mitochondrial transporter inhibitor.

[0096] In cases where the MIRO1 status indicates the presence of Parkinson's disease, in some embodiments, the Parkinson's disease is a PTEN-induced putative kinase 1 (PINK-1)-associated form of PD. In some cases, the Parkinson's disease is associated with a mutation in any of Parkin, leucine-rich repeat kinase 2, alpha-synuclein, parkinsonin 7, 13A2-type ATPase, group VI phospholipase A2, DnaJ (Hsp40) homolog subfamily C member 6, eukaryotic translation initiation factor 4γ-1, F-box protein 7, GRB10 interacting GYF protein 2, HtrA serine peptidase 2, synaptobrevin 1, or vacuolar protein sorting protein 35 homolog. The Parkinson's disease may be a sporadic form of Parkinson's disease.

[0097] The MIRO1 reducing agent can be administered alone or in combination with any pharmaceutically acceptable carrier or salt known in the art as described below.

[0098] The method for detecting MIRO1 levels can be any method known in the art. In some embodiments, the method for detecting MIRO1 levels comprises electrophoresis, chromatography, enzyme assay, binding assay or a combination thereof. In some embodiments, the method for detecting MIRO1 levels comprises ELISA. In some embodiments, the method comprises the assay described in any one of the examples.

[0099] As described herein, MIRO1 status is associated with the presence of certain neurodegenerative disorders or an increased risk of developing certain neurodegenerative disorders. In some embodiments, elevated MIRO1 levels are associated with neurodegenerative disorders (e.g., Parkinson's disease). In some embodiments, the MIRO1 level in the subject is about 5% or more, about 10% or more, or about 20% or more relative to the control MIRO1 level in the control subject, for example, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, or about 80% or more, for example, about 90% or more, about 95% or more, about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 800% or more, about 1000% or more, about 2000% or more, about 3000% or more. In some embodiments, the MIRO1 level in the subject is about 20% or more relative to the control MIRO1 level in the control subject. In some embodiments, the MIRO1 level in a subject is about 30% or more higher relative to the control MIRO1 level in a control subject.

[0100] The subject may be experiencing symptoms of a neurodegenerative disorder. In one aspect, the invention provides a method for diagnosing whether a subject has a neurodegenerative disorder, comprising: (a) obtaining a MIRO1 level in a subject; (b) comparing the MIRO1 level with information from a control, wherein the information is predetermined to indicate the absence of the neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Parkinson's disease.

[0101] Movement disorders. Movement disorders are usually classified as either reduced or slow movements (hypokinesia) or increased movements (hyperkinesia). The classic and most common movement disorder is Parkinson's disease. Hyperkinesias include tremors, myoclonus, dystonia, chorea, and tics.

[0102] Atypical Parkinson's syndrome refers to a group of neurodegenerative disorders other than Parkinson's disease that have certain characteristics of Parkinson's disease but also have certain different clinical features and different pathologies. As shown herein, the Miro1 state of the subject evaluated by the method described herein can be used to distinguish PD from atypical Parkinson's syndrome. Atypical Parkinson's syndrome covers neurodegenerative disorders such as progressive supranuclear palsy, Lewy body dementia, corticobasal ganglia degeneration and multiple system atrophy. Defects suggesting neurodegenerative disorders other than Parkinson's disease include gaze palsy, signs of corticospinal tract dysfunction (e.g., hyperreflexia), myoclonus, autonomic dysfunction (if early or severe), cerebellar ataxia, obvious dystonia, ideomotor apraxia (inability to imitate hand movements), early dementia, early falls and wheelchair use.

[0103] Parkinson's disease. Parkinson's disease (PD), also known as idiopathic or primary parkinsonism, hypokinesia-rigidity with tremor syndrome / HRS, or parkinsonism, is a degenerative disease of the central nervous system. The motor symptoms of Parkinson's disease are caused by the death of dopamine-producing neurons in the substantia nigra (an area of ​​the midbrain) and putamen; the cause of this cell death is not yet clear. Early in the course of the disease, the most obvious symptoms are those related to movement; these include tremors, rigidity, resting tremor, bradykinesia, postural instability, slow movements, and difficulty walking and gait problems. Later, thinking and behavioral problems may develop with dementia, such as cognitive impairment, hallucinations, delusions, abnormal behavior, depression, and sleep and arousal disorders, usually in the later stages of the disease, with depression being the most common psychiatric symptom. Other symptoms include problems with sensation (loss of smell), sleep (sleep and arousal disorders), and mood problems, constipation, low blood pressure, frequent urination, impotence, and sweating. Parkinson's disease is more common among older people, with most cases occurring after age 50.

[0104] Parkinson's disease can be either familial or sporadic. Familial means that the disease is inherited, i.e., heritable gene mutations are passed from parent to offspring through gametes. Many different heritable mutations are associated with PD, including, for example, PTEN-induced putative kinase 1 (PINK-1); Parkin (also known as RBR Mutations in E3 ubiquitin-protein ligase, or PARK2); leucine-rich repeat kinase 2 (LRRK2); alpha-synuclein (SNCA, PARK4); ubiquitin carboxyl-terminal hydrolase L1 (UCHL1); parkinson protein 7 (PARK7, DJ-1); type 13A2 ATPase (ATP13A2); group VI phospholipase A2 (PLA2G6); DnaJ (Hsp40) homolog subfamily C member 6 (DNAJC6, PARK19); eukaryotic translation initiation factor 4 gamma-1 (EIF4G1, PARK18); F-box protein 7 (FBXO7); GRB10-interacting GYF protein 2 (GIGYF2); HtrA serine peptidase 2 (HTRA2); synaptobrevin 1 (SYNJ1); or vacuolar protein sorting protein 35 homolog (VPS35). The sporadic form means that the disease occurs sporadically, ie, due to sporadic mutations in a gene, such as one of the genes listed above.

[0105] In some cases, the subject does not experience symptoms of a neurodegenerative disorder. In one aspect, the invention provides a method for detecting whether a subject is at increased risk of developing a neurodegenerative disorder, comprising: (a) obtaining a MIRO1 level in a subject; (b) comparing the MIRO1 level with information from a control, wherein the information is predetermined to indicate the absence of the neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is Parkinson's disease.

[0106] In some embodiments, a method for diagnosing whether a subject has a neurodegenerative disorder comprises: providing a sample from a subject suspected of having a neurodegenerative disorder; determining the MIRO1 level in the sample; comparing the determined MIRO1 level with a control MIRO1 level; detecting an increase in the MIRO1 level compared to the control MIRO1 level in the sample from the subject; and diagnosing the presence of a neurodegenerative disorder in the subject with an increased MIRO1 level. After the neurodegenerative disorder is diagnosed, the subject can be treated with a method described herein or known in the art. In some embodiments, the method comprises an assay as described in any one of the examples.

[0107] Method for determining Miro1 status

[0108] The present invention provides an assay for determining the state of Miro1 in a cell or cell population. Miro1 is usually removed from damaged (e.g., depolarized) mitochondria to facilitate its clearance (clearance by mitochondrial autophagy), but this degradation may be defective, and this defect can be detected using a biochemical assay. The degradation defect is closely related to PD or susceptibility to PD. After depolarization, a considerable proportion of Parkinson's disease subjects have insufficient Miro1 removal. However, in control cells, Miro1 can still be effectively degraded after depolarization. Detection of this PD-related defect can be used for PD diagnosis and PD susceptibility prognosis, optionally including steps for treating subjects so diagnosed; in clinical trials, etc., monitoring the clinical response to PD after treatment; screening drugs for efficacy in reducing this Miro1 degradation defect, etc. These methods can accurately assess Parkinson's disease in an early and clinically practical manner.

[0109] The analytical method can be performed by examining the presence of Miro1 polypeptides in cellular components after mitochondrial uncoupling (including chemically induced depolarization). These assays can be performed using cell lines, cells obtained from subjects (including but not limited to biological samples such as fibroblasts, peripheral blood lymphocytes, etc.). The assay is usually performed on viable cells (i.e., living cells). Fibroblasts are a suitable source of cells in an individual and can be easily obtained from a subject through minimally invasive, painless surgery. Cultured cells can be derived from patient or control samples; and can be modified to obtain genetically modified cells, in vitro differentiated cells, cells exposed to candidate therapeutic agents; and the like. In some embodiments, a cell population from a subject is assayed to determine the Miro1 phenotype of the individual.

[0110] In addition, the present invention can use cells that have been genetically altered to gain or lose genetic function by, for example, transfection or transduction with a recombinant gene, or by using antisense technology. Methods for forming genetically modified cells are known in the art, see, for example, "Molecular Biology: A Laboratory Manual", Ausubel et al., eds., John Wiley & Sons, New York, NY, 2000. The genetic alteration can be a gene knockout, typically resulting from homologous recombination, thereby disrupting expression of the targeted gene, or a gene knockin, in which a genetic sequence that is not normally present in the cell is stably introduced.

[0111] The present invention can use a variety of methods to achieve gene knockout, including site-specific recombination, antisense or dominant negative mutation expression, etc. For gene targeting, gene knockout can cause partial or complete loss of function of one or two alleles in the endogenous gene. Preferably, the expression of the targeted gene product is undetectable or insignificant in the analyzed cell. This can be achieved by destroying the coding sequence, such as inserting one or more stop codons, inserting DNA fragments, etc., the disappearance of the coding sequence, the replacement of the coding sequence by the stop codon, etc. In some cases, the introduced sequence can be finally deleted from the genome, so that the original sequence is changed in a net manner.

[0112] The cell sample can contain, for example, at least about 10 2 cells, at least about 10 3 cells, at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 Cells or more. Optionally, more cells are measured by multiple aliquots. The cells are contacted with an agent that uncouples or depolarizes mitochondria. The concentration and exposure time of the agent are sufficient to uncouple or depolarize the mitochondria. For example, a CCCP concentration of about 10-100 μM (e.g., about 20 to 80 μM, about 30 to 50 μM) is sufficient. The dosage of other agents can provide activity comparable to these CCCP concentrations. The cells are incubated for a sufficiently long time to depolarize the mitochondria and begin to clear, for example, at least about 1 hour, at least about 2 hours, usually no more than about 24 hours, and can be about 1 to 24 hours, about 2 to 20 hours, about 3 to 18 hours, about 4 to 14 hours, about 5 to 10 hours, and can be about 4 to 8 hours.

[0113] In some embodiments, the agent is a mitochondrial-specific uncoupler, such as a proton carrier. Agents suitable for this purpose include FCCP, CCCP, DNP, BAM15, etc., known in the art and as described herein. After mitochondrial depolarization, the cells are lysed and the Miro1 level is assessed, wherein the Miro1 degradation defect relative to the control indicates that it is associated with PD. The defect can be expressed as, for example, the ratio of the Miro1 level in the cell lysate contacted with the mitochondrial uncoupler relative to the same cell in the absence of the uncoupler. The Miro1 present in PD-related cells can be at least 2 times, at least 3 times, at least 4 times or more that of normal cells.

[0114] Assess whether there is a change in Miro1 degradation in response to mitochondrial uncoupling in a test sample from a subject. The term "change" in polypeptide level as used herein refers to a change in the corresponding level compared to a control sample. The control sample is a sample corresponding to the test sample (e.g., from the same type of cell) and is from a subject not affected by PD susceptibility. Changes in polypeptide levels in the test sample compared to the control sample indicate susceptibility to PD. Protein levels can be measured by a variety of methods, including enzyme-linked immunosorbent assay (ELISA), western blotting, immunoprecipitation, and immunofluorescence.

[0115] For example, in one aspect, an antibody that binds to the polypeptide (e.g., as described above) can be used for capture or detection. The antibody can be a polyclonal antibody, or more preferably a monoclonal antibody. A complete antibody or a fragment thereof (e.g., Fab or F(ab')) can be used. 2 ). With respect to a probe or antibody, the term "labeled" is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, enzyme-linked assays, radiolabeled antibodies, and the like as known in the art.

[0116] Miro1 degradation can be monitored in a variety of ways. Western blot analysis using antibodies that specifically bind to Miro1 can be used to identify the presence of Miro1 in a test sample, such as after cell fractionation, and the analysis can be quantitative. In other embodiments, high-throughput immunoassays are preferred. For convenience, the removal of Miro1 in a subject sample is detected by immunoassay (e.g., ELISA or other high-throughput affinity assays).

[0117] In some embodiments, the Miro1 assay as described above is used for the diagnosis and clinical monitoring of movement disorders, including but not limited to Parkinson's disease. In some embodiments, the methods of the present invention are used to determine the efficacy of therapies for treating movement disorders, such as in vitro (e.g., drug screening assays, etc.), at the subject level, in subject group analysis (e.g., in the form of clinical trials), etc. Clinical trial embodiments may involve comparisons of two or more time points in a subject or subject group. Since the subject receiving treatment is given a therapeutic agent, a treatment regimen is implemented, or the subject is stimulated with a disease-inducing agent, it is expected that the patient's state between the two time points will be different. The response of a subject with movement disorders to treatment is assessed by detecting the ability of a cell sample (including but not limited to a fibroblast sample) from the subject to degrade Miro1 after mitochondrial damage (e.g., mitochondrial depolarization).

[0118] In some embodiments, the method comprises identifying a subject as having PD or being susceptible to PD, such as according to the above-described Miro1 degradation criteria; administering a dose of a therapeutic agent to the patient, and quantifying Miro1 degradation in response to mitochondrial depolarization in at least one patient sample.

[0119] In some embodiments, the methods of the invention are used to determine the efficacy of a therapy for treating a movement disorder at the subject level or in a subject group analysis (e.g., in a clinical trial format). Such embodiments generally involve a comparison of two time points in a subject or subject group. Due to the administration of a therapeutic agent, the implementation of a treatment regimen, or the disease challenge to the subject being treated, the patient's state is expected to be different between the two time points.

[0120] For regulatory approval of treatment regimens that provide symptomatic benefit in subjects with Parkinson's disease, clinical trials have used double-blind, placebo-controlled, parallel-group designs with fixed or flexible dosing strategies. Treatment efficacy has been assessed using a variety of efficacy outcome measures (one or more combinations of subscales of the Unified Parkinson's Disease Rating Scale [UPDRS]) as well as the need for additional symptomatic treatment (e.g., dopaminergic agonists, levodopa).

[0121] Exemplary assays for MIRO1 quantification include those described in the following publications: Hsieh CH, Li L, Vanhauwaert R, Nguyen KT, Davis MD, Bu G, et al., Miro1 marks a Parkinson's disease subpopulation and Miro1 reduction prevents neuronal loss in a Parkinson's disease model, Cell Metabolism, 2019;1131-1140; and Shaltouki A, Hsieh C-H, Kim MJ, Wang X, α-synuclein delays mitophagy and targeting Miro prevents neuronal loss in a Parkinson's disease model, Acta Neuropathologica Sinica, 2018;136:607-620.

[0122] Therapeutic method

[0123] The present invention provides a method for treating neurodegenerative disorders, comprising administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro (e.g., Miro1) in Parkinson's disease cells having depolarized mitochondria to an amount that is equivalent to the amount of Miro in control healthy cells having depolarized mitochondria that have not been contacted with the Miro reducing agent or within 5%, within 10%, within 15%, within 20%, within 25%, within 30%, within 35%, within 40%, within 45%, within 50%, within 55%, within 60%, within 65% or within 70% of the amount of Miro in the control cells.

[0124] The neurodegenerative disorders included in the method of the present invention include, but are not limited to, neurological diseases with symptoms similar to those observed in Parkinson's disease-related disorders. In some cases, the neurological disease may exhibit symptoms similar to Parkinson's disease, atypical Parkinson's disease, or Parkinson's superposition disease. Examples include, but are not limited to, drug-induced Parkinson's syndrome, progressive supranuclear palsy, vascular Parkinson's syndrome, Lewy body dementia, diffuse Lewy body disease, corticobasal ganglia degeneration, multiple system degeneration (Shay-De syndrome), Alzheimer's disease, Pick's disease, and progressive supranuclear palsy (Steel-Richardson syndrome). Other disorders also included in the method of the present invention include age-related dementia and other dementias and symptoms showing memory loss (including vascular dementia), diffuse white matter disease (Binswanger's disease), endocrine or metabolic dementia, head trauma and diffuse brain injury dementia, boxing dementia, and frontal lobe dementia. In some cases, the neurological disease may not respond well to dopaminergic therapy and may be caused by a variety of vascular, drug-related, infectious, toxic, structural, and other known secondary causes. Drug-induced parkinsonism may be caused by agents that block postsynaptic dopamine D2 receptors with high affinity, such as antipsychotics and antiemetics as well as sodium valproate, antidepressants, reserpine, tetrabenazine, etc.

[0125] A variety of subjects are suitable for treatment with agents identified by the methods of the invention. Suitable subjects include any subject who exhibits symptoms of Parkinson's disease, such as bradykinesia; repetitive movements; tremors; limb rigidity; gait and balance problems; inability to aim at the bull's eye due to eye muscle weakness; weakness; sensory loss; non-motor manifestations such as REM sleep behavior disorder; neuropsychiatric symptoms (including mood disorders and cognitive changes); anxiety; apathy; changes in thinking ability; changes in attention or alertness level and hallucinations; intellectual and functional decline; amnesia; personality changes; autonomic dysfunction affecting cardiovascular, respiratory, genitourinary, gastrointestinal and sweating functions; breathing and swallowing difficulties; inability to sweat; orthostatic hypotension; pain; constipation and loss of smell. In some cases, the subject may have significant speech or language disorders, significant frontal lobe symptoms and freezing of gait.

[0126] In certain embodiments, the subject may not exhibit any obvious symptoms of Parkinson's disease. In some cases, the subject in need thereof may exhibit increased susceptibility to infection, hypothermia, weaker bones, stiff joints, arthritis, hunched posture, slowed movements, decreased overall energy, constipation, urinary incontinence, memory loss, slower thinking, slower reflexes, difficulty with balance, decreased vision, decreased peripheral vision, hearing loss, wrinkling of the skin, graying of hair, weight loss, and loss of muscle tissue.

[0127] In some cases, the subject can be a person who has been diagnosed with Alzheimer's disease; a subject who has had one or more strokes; a subject who has suffered a traumatic head injury; an individual with high serum cholesterol levels; a subject with a proteinopathy (including deposits in brain tissue); a subject who has had one or more cardiac events; a subject who has undergone cardiac surgery; and a subject with multiple sclerosis.

[0128] In some cases, the subject may exhibit symptoms associated with a neurological disease, including motor neuron diseases such as amyotrophic lateral sclerosis, degenerative ataxias, corticobasal degeneration, ALS-Parkinson's disease-dementia complex of Guam type, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease / spinocerebellar ataxia type 3 and olivo-ponto-cerebellar degeneration, Gilles De La Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff's disease, familial spastic disease, Wohlfart-Kugelberg-Welander disease, spastic paralysis, progressive multifocal leukoencephalopathy, and prion diseases (including Creutzfeldt-Jakob disease, Gerstmann-Straussner disease, Kuru disease, and fatal familial insomnia). Also included are other neurodegenerative disorders caused by cerebral ischemia or infarction, including embolic occlusions and thrombotic occlusions, and any type of intracranial hemorrhage (including, but not limited to, epidural, subdural, subarachnoid, and intracerebral), as well as intracranial and intraspinal lesions (including, but not limited to, contusions, penetrations, shearing, compressions, and lacerations). In some cases, comparison of Miro levels in cells from a subject after mitochondrial depolarization to Miro levels in cells from a control healthy subject after mitochondrial depolarization can be used as a diagnostic assay to identify subjects who may benefit from prophylactic use of the Miro reducing agent to prevent distortion of mitochondrial homeostasis. Mitochondrial homeostasis refers to the balance between mitochondrial biogenesis, mitophagy, trafficking, fission, fusion, and other processes that maintain mitochondrial function and that rely at least in part on Miro function.

[0129] Mitochondrial depolarization used in the present invention refers to the process in which the potential difference on the mitochondrial membrane decreases from the steady-state level, and the mitochondrial membrane potential changes from the resting potential to the depolarized direction, and from negative to positive. The voltage gradient on the inner mitochondrial membrane is partially affected by the mitochondrial permeability transition pore. In some cases, depolarization below a certain Δψm may indicate impaired mitochondrial function and may initiate mitochondrial autophagy. In some cases, mitochondrial depolarization may precede the translocation of proteins such as Parkin and Pink1. In some cases, cytochrome may be released accompanied by mitochondrial depolarization. Mitochondrial depolarization can be detected by Rhodamine 123, Mitotracker Red, DiOC 6 or tetramethylrhodamine methyl ester (TMRE), methyl ester (TMRM), nonylacridine orange (NAO), SaphranineO, merycyanine 540, JC-1 or JC-9, etc. Mitochondrial depolarization can be accompanied by a decrease in mitochondrial resting potential (as measured by quantitative assay) of at most 5%, at most 7%, at most 10%, at most 15%, at most 18%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 90%, at most 95% or at most 100%.

[0130] Depolarizing agents can induce mitochondrial depolarization. Mitochondrial depolarizing agents can be calcium dysregulation, ROS production, chemicals such as barbiturates, ginsenoside-Rh2, rotonene, complex-I inhibitors, complex-II inhibitors, complex-III inhibitors, complex IV inhibitors, mycotoxins such as auroverticin AE, limonostigmine A and B, staurosporin and oxamycin, efrapeptin, oligomycin AD, vancomycin, antimycins, naturally occurring flavonoids, propranolol, local anesthetics, herbicides paraquat, pyrethroids, DDT, parathion, diethylstilbestrol, several cationic dyes and organotin compounds, uncouplers such as substituted phenols, carbonyl cyanide 4-(triazine)-4-nitropropene. fluoromethoxy)phenylhydrazone (FCCP), carbonyl cyanide m-chlorophenylhydrazone (CCCP), trifluoromethylbenzimidazole, salicylanilide and carbonyl cyanide phenylhydrazone, endogenous and exogenous free fatty acids (FFA) and fatty acid compounds such as perfluorodecanoic acid, sulfamide and methyl-substituted hexadecanoic acid, peptides such as adenine nucleotide translocase, ion carriers such as gramicidin (gramicidin A, D and S), nigericin, valinomycin, cationic uncouplers such as cyanine dye tri-S-C4(5), Cu2`-(o-phenanthroline)2 complex, pentamidine, membrane active peptides such as alamethicin, wasp toxin, alternative electron acceptors such as doxorubicin, paraquat and various substituted naphthoquinones and nitrosamines.

[0131] In one aspect, the present invention provides a method for measuring Miro degradation in cells obtained from subjects diagnosed with sporadic or familial neurodegeneration under conditions where the mitochondrial membrane permeability of the cells is changed. In some cases, the cells are fibroblasts derived from tissues obtained from subjects diagnosed with sporadic or familial neurodegeneration, and the method comprises obtaining tissue from the patient, preferably a skin biopsy, cutting the skin biopsy slices into slices of uniform size, transferring the cut skin biopsy slices to a tissue culture plate coated with gelatin, and replacing the culture medium with complete DMEM / 20% FBS culture medium every 2-3 days until the fibroblasts are confluent. In some cases, the fibroblasts are obtained from subjects diagnosed with sporadic or familial forms of Parkinson's disease. In some cases, the neurodegenerative disorder is Parkinson's disease or a Parkinson-like disease. In some cases, the subject suffers from the disease and is asymptomatic. In some cases, the subject has risk factors for the disease and is asymptomatic. The term sporadic Parkinson's disease used herein refers to a non-familial form of Parkinson's disease. In some cases, sporadic PD is caused by environmental factors. In some cases, subjects with sporadic PD may carry genes associated with PD that have low penetrance or a later age of onset, making the occurrence of any familial events less obvious.

[0132] In some cases, the cell is a subject-specific induced pluripotent stem cell (iPSC) associated with Parkinson's disease or Parkinson's disease-like disease. In one embodiment, the induced pluripotent stem cell is derived from human fibroblasts. In some cases, the subject may suffer from a genetic form of the disease or a disseminated form of the disease. In a related embodiment, the induced pluripotent stem cell is produced without the use of a retrovirus or a lentivirus. In a specific embodiment, the induced pluripotent stem cell is produced using a method comprising using three factors (e.g., OCT4, SOX2, and KLF4). In another embodiment, the induced pluripotent stem cell is further differentiated to adapt to the midbrain dopaminergic cell fate. In some cases, within about 20 days, the induced pluripotent stem cell differentiates to adapt to the cell fate.

[0133] In some embodiments, the cell is a neuronal cell derived from a subject diagnosed with Parkinson's disease or a Parkinson-like disease, and the method comprises obtaining fibroblasts from the subject, dedifferentiating the fibroblasts into pluripotent stem cells, and differentiating the stem cells toward a neuronal cell fate. In one embodiment, the fibroblasts are dermal fibroblasts. In another embodiment, the stem cells differentiate toward a midbrain dopaminergic cell fate. In a specific embodiment, the dedifferentiation of the fibroblasts induces pluripotency.

[0134] In some embodiments, the subject carries a genetic variation or mutation known to be associated with Parkinson's disease or Parkinson's-like disease. In other embodiments, the target genetic variation is a copy number variation of the target gene. In a specific embodiment, the cell line has three copies of the target gene. The genetic variation or mutation can be a deletion, insertion, complex polymorphic variant, deletion, substitution, conversion, transversion or duplication of one or more nucleotides in the target gene. In an exemplary embodiment, the target gene is selected from PARK1 (SNCA or α-synuclein), PARK2 (Parkin), PARK5 (UCHL1), PARK6 (PINK1), PARK7 (DJ-1), PARK8 (LRRK2) and PARK 11 (GIGFY2). In a specific embodiment, the target gene is PARK1 (SNCA or α-synuclein). In other specific embodiments, the target gene is PARK8 (LRRK2). In one embodiment, the subject carries a homozygous mutation of LRRK2, and the mutation comprises a G2019S mutation. In some embodiments, the subject suffers from Parkinson's disease. In one embodiment, the subject suffers from an idiopathic form of Parkinson's disease.

[0135] In some aspects, the method includes providing a cell with a sequence variation or multiple copies of a target gene, and inducing pluripotency, multipotency or omnipotence in the cell to prepare a cell line with a target gene variation. The method may also include identifying a subject with a target gene variation, and obtaining one or more cells from the subject. The subject-derived cell may be a fibroblast, a tumor cell, a bone marrow cell, a gastric cell, a blood cell (e.g., a leukocyte, a blood progenitor cell), a hepatocyte, etc., or any suitable or related source cell to be obtained from the subject. The method for producing a cell line with a target gene copy number variation may also include inducing differentiation of the cell line. The cell line may be differentiated into any target cell type, including endoderm, ectoderm, mesoderm, such as nerves, such as neuronal cell lines, epithelial cell lines, myocardial cell lines, etc.

[0136] In some aspects, the cell lines and methods of use thereof include cell lines having genetic copy number variation, wherein the variation is at least one copy of a gene, such as two or three copies. In other aspects, the copy number variation is one or more deletions, insertions, or complex polymorphic variants of a gene of interest.

[0137] In some aspects, the cell lines and methods of using the same include cell lines having genetic variations that are mutations of a target gene. For example, the mutation can be a deletion, substitution, conversion, transversion or duplication of one or more nucleotides in the target gene. In some embodiments, the mutation is a point mutation.

[0138] In one aspect, the invention provides a method of treating a neurodegenerative disorder comprising administering a Miro1 reducing agent to a subject in need thereof, wherein the Miro1 reducing agent reduces Miro1 in sporadic Parkinson's disease cells according to an assay comprising the following steps:

[0139] (a) Fibroblasts from subjects with sporadic Parkinson's disease are seeded into wells of an array;

[0140] (b) 24 hours after step (a), adding the candidate agent to the first test well, while not adding the candidate agent to the first control well;

[0141] (c) 10 hours after step (b), adding FCCP to the first test well and the first control well;

[0142] (d) 14 hours after step (c), fixing the cells in the first test well and the first control well with ice-cold 90% methanol; and

[0143] (e) immunostaining the cells in the first test well and the first control well with anti-Miro1 and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and imaging with a confocal microscope to measure the Miro1 intensity / cell for the images of the first test well and the first control well.

[0144] In some cases, the Miro reducing agent reduces Miro1 in the first test well by up to three, up to four, up to five, up to six or up to seven standard deviations relative to the first control well. In some cases, the Miro reducing agent reduces Miro1 in the first test well by about 2 to about 7, about 2 to about 5, about 2 to 4 or about 2 to 6 standard deviations relative to the first control well. In some cases, the candidate agent reduces Miro1 in the second test well by less than one or less than half a standard deviation relative to the second control well, and the candidate agent is a Miro reducing agent. In some cases, Parkinson's disease cells are obtained from familial Parkinson's disease subjects. In some cases, the Miro level in the Parkinson's disease cells is increased compared to cells from healthy subjects. In some cases, the Parkinson's disease cells show Miro levels comparable to those of healthy subjects. In some cases, the mitochondrial depolarizing agent is not FCCP. In some embodiments, the Miro reducing agent is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule or an aptamer. In some cases, the Miro is detected by ELISA, Western blotting, or fluorescent real-time imaging.

[0145] In one aspect, the invention provides a method of treating a neurodegenerative disorder comprising administering a Miro1 reducing agent to a subject in need thereof, wherein the Miro1 reducing agent reduces Miro1 in sporadic Parkinson's disease cells as determined by:

[0146] (a) Fibroblasts from subjects with sporadic Parkinson's disease are seeded into wells of an array;

[0147] (b) 24 hours after step (a), adding the candidate agent to the first test well, while not adding the candidate agent to the first control well;

[0148] (c) 10 hours after step (b), adding FCCP to the first test well and the first control well;

[0149] (d) 14 hours after step (c) is completed, the cells in the first test well and the first control well are fixed with ice-cold 90% methanol; and (e) the cells in the first test well and the first control well are immunostained with anti-Miro1 and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and imaged with a confocal microscope to measure the intensity of Miro1 / cell for the images of the first test well and the first control well. In one aspect, the present invention provides a method for treating a neurodegenerative disorder, comprising administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro1 in the sporadic Parkinson's disease cells with depolarized mitochondria by two, three, four, five or six standard deviations or more relative to the sporadic Parkinson's disease cells with depolarized mitochondria that are not treated with the Miro reducing agent.

[0150] In some cases, the Miro reducing agent identified in the above assay does not reduce Miro1 in sporadic Parkinson's disease cells as determined by:

[0151] (a1) Fibroblasts from a subject with sporadic Parkinson's disease are seeded into the wells of an array;

[0152] (b1) 24 hours after step (a1), adding the candidate agent to the second test well, while not adding the candidate agent to the second control well;

[0153] (c1) 14 hours after step (b1), fixing the cells in the second test well and the second control well with ice-cold 90% methanol; and

[0154] (d1) immunostaining the cells in the second test well and the second control well with anti-Miro1 and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and imaging with a confocal microscope to measure the intensity of Miro1 / cell for the images of the second test well and the second control well. In one aspect, the present invention provides a method for treating a neurodegenerative disorder, comprising administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro1 in the sporadic Parkinson's disease cells with depolarized mitochondria by two, three, four, five or six standard deviations or more relative to the sporadic Parkinson's disease cells with depolarized mitochondria not treated with the Miro reducing agent, and the Miro reducing agent reduces Miro in the sporadic Parkinson's disease cells by less than one standard deviation relative to the sporadic Parkinson's disease cells not treated with the Miro reducing agent.

[0155] In one aspect, the present invention provides a method for treating a neurodegenerative disorder, comprising administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria. In some cases, the Miro reducing agent reduces Miro1 in Parkinson's disease cells having depolarized mitochondria to an amount that is comparable to the amount of Miro in control healthy cells having depolarized mitochondria that are not contacted with the Miro reducing agent or within 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62.%, 65%, 67.5%, 70%, 72.5%, 75% of the amount of Miro in the control cells. In some cases, the Miro reducing agent reduces Miro in Parkinson's disease cells to an amount between 15%-25%, 15%-30%, 20%-30%, 20%-40%, 25%-40%, 25%-35%, 25%-45%, 30%-45%, 30%-50%, 35%-55%, 35%-60%, 35%-50%, 35%-55%, 40%-50%, 40%-55%, 40%-60%, 45%-65%, 45%-70%, 45%-75% of the amount of Miro in control healthy cells with depolarized mitochondria that are not contacted with the Miro reducing agent. In some cases, the healthy cells with depolarized mitochondria are healthy cells contacted with a mitochondrial depolarizing agent.

[0156] In one aspect, the present invention provides a method for treating a neurodegenerative disorder, comprising administering a Miro-reducing agent to a subject in need thereof, wherein the Miro-reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that have not been contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by three standard deviations or more compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that have not been contacted with the Miro-reducing agent. In some cases, the Miro-reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by two standard deviations or more compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that have not been contacted with the Miro-reducing agent. In some cases, the Miro reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by four standard deviations or more compared to a reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that were not contacted with the Miro reducing agent. In some cases, the Miro reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by five standard deviations or more compared to a reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that were not contacted with the Miro reducing agent.

[0157] In one aspect, the present invention provides a method for treating a neurodegenerative disorder, comprising administering a Miro1 reducing agent to a subject in need thereof, wherein (a) the Miro1 reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by two standard deviations or more compared to the reduction in Miro1 in control Parkinson's disease cells with depolarized mitochondria that have not been contacted with the Miro1 reducing agent; and (b) the Miro1 reducing agent reduces Miro1 in Parkinson's disease cells with non-depolarized mitochondria by one standard deviation or less compared to the reduction in Miro1 in control Parkinson's disease cells with non-depolarized mitochondria that have not been contacted with the Miro1 reducing agent. In some cases, the Miro1 reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by three standard deviations or more compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that have not been contacted with the Miro1 reducing agent. In some cases, the Miro reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by four standard deviations or more compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that have not been contacted with the Miro reducing agent. In some cases, the Miro reducing agent reduces Miro1 in Parkinson's disease cells with depolarized mitochondria by five standard deviations or more compared to the reduction in Miro1 in control depolarized sporadic Parkinson's disease cells with depolarized mitochondria that have not been contacted with the Miro reducing agent. In some cases, the Parkinson's disease cells with depolarized mitochondria are Parkinson's disease cells contacted with a mitochondrial depolarizing agent. In some cases, the Parkinson's disease cells with depolarized mitochondria are Parkinson's disease cells that have not been contacted with a mitochondrial depolarizing agent. In some cases, the Parkinson's disease cells are sporadic Parkinson's disease cells. In some cases, the Parkinson's disease cells are familial Parkinson's disease cells. In some cases, the Parkinson's disease cells are fibroblasts. In some cases, the Miro levels in the Parkinson's disease cells are increased compared to cells from healthy subjects. In some cases, the Parkinson's disease cells exhibit Miro levels comparable to healthy subjects. In some cases, the mitochondrial depolarizing agent is not FCCP. In some embodiments, the Miro reducing agent is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule or an aptamer. In some cases, the Miro is detected by ELISA, Western blotting or fluorescent real-time imaging.

[0158] In some cases, the subject has a higher Miro level, e.g., a higher Miro1 level, compared to the corresponding Miro level in a control subject. For example, the Miro1 level in the subject may be about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 1000% or more than the control subject. In some cases, the Miro1 level of the subject may be about 20% or more or about 30% or more higher than the Miro1 level in the control subject.

[0159] In some cases, the candidate agent is a peptide, antibody, protein, protein fragment, aptamer or small molecule. In one embodiment, the screening assay of the present invention is a high-throughput or ultra-high-throughput assay. For example, the screening assay of the present invention can be performed in a multi-well format, such as a 96-well, 384-well or 1,536-well format, and is suitable for automation. In detail, each well of a microtiter plate can be used to individually assay a selected test agent. In some cases, it is necessary to observe the concentration or incubation time effects of a single test, and a single test agent can be tested every 5-10 wells. Multiple plates can be assayed per day; using this method, assays screen up to about 6,000, 20,000, 50,000 or more than 100,000 different compounds.

[0160] Candidate agents can be proteins or protein fragments, antibodies or antibody fragments, small molecules or aptamers. The candidate agents can be in the form of candidate agent libraries, such as combinations or randomized libraries that provide sufficient diversity ranges. Candidate agents are optionally connected to fusion partners, such as targeting compounds, markers or detectable moieties, rescue compounds, dimerization compounds, stable compounds, addressable compounds and other functional moieties. In some cases, the candidate agents are attached to the surface of the array. In some cases, the candidate agents are delivered to cells expressing Miro in the form of liquid samples.

[0161] In some cases, the Parkinson's disease cells and healthy cells express Miro protein linked to a marker or reporter gene moiety. A "marker" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful markers include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxin or haptens, and proteins such as those that can be made into detectable forms by incorporating radioactive markers into the peptides or for detecting antibodies that specifically react with the peptides. In some cases, the Miro level can be detected by using fluorescence, luminescence, chemiluminescence, absorbance and other optical methods. In some cases, the difference in Miro levels between the Parkinson's disease cells and healthy cells is measured by a computer algorithm.

[0162] In some cases, the cell can be genetically engineered to express Miro protein labeled as an enzyme reporter gene, which can generate a fluorescent signal or can be combined with a small molecule that can be labeled with a fluorescent part. In some examples, the small molecule can be labeled with a fluorescent reagent such as fluorescein, rhodamine, TexasRed, BODIPY and other commercially available molecules (e.g., molecules available from Molecular Probes / Invitrogen and other suppliers). A variety of fluorescence readings can be generated. In other examples, ligands and other probes can be directly labeled with fluorescein or other fluorophores to detect binding to cell proteins; or can be labeled with enzymes such as alkaline phosphatase or horseradish peroxidase to achieve indirect detection and positioning of signals. In other examples, Miro can be labeled with an enzyme, which can be used to generate a fluorescent signal in a living cell by using a cell-permeable specific substrate, which becomes a fluorescent form or shifts its fluorescence spectrum when the enzyme is cleaved, thereby shifting the absorption or emission wavelength of the fluorophore. In other embodiments, Miro can be labeled with an enzyme that can be used to generate a fluorescent signal in living cells by cleaving a covalent assembly of an emission-absorption matched fluorophore pair that maintains a resonance energy transfer between the two fluorophores in a covalently assembled form that is lost when the two fluorophores are separated.

[0163] Luminescent, fluorescent, or bioluminescent signals can be easily detected and quantified using any of a variety of automated and / or high-throughput instrumentation systems, including fluorescence multiwell plate readers, fluorescence-activated cell sorters (FACS), and automated cell-based imaging systems that provide spatial resolution of the signal. A variety of automated HCS instrumentation systems have been developed, including automated fluorescence imaging and automated microscopy systems developed by Cellomics, Amersham, TTP, Q3DM, Evotec, Universal Imaging, and Zeiss. Fluorescence recovery after photobleaching (FRAP) and time-lapse fluorescence microscopy techniques have also been used to study protein mobility in living cells.

[0164] On the one hand, the present invention also provides a method for identifying a diagnostic cell phenotype, the method comprising comparing a cell group from a subject with a cell from a subject who does not suffer from Parkinson's disease, wherein the cell phenotype detected is Miro degradation after mitochondrial depolarization. In some cases, Miro levels are compared on a computer. The cell can be a fibroblast. The cell can be an induced pluripotent stem cell, a cell differentiated into a neural stem cell from an induced stem cell, or a neuron. In one embodiment, the detected response is a change in mitochondrial function, mitochondrial fission, fusion, morphology, mitochondrial autophagy, mitochondrial transport, intracellular calcium levels, or other cell characteristics that depend on Miro function.

[0165] In one aspect, the present invention also relates to a method for determining the risk of a subject suffering from Parkinson's disease, the method comprising comparing at least one phenotype determined in a first group of cells derived from the subject with at least one phenotype determined in a second group of cells derived from a subject not suffering from Parkinson's disease and at least one phenotype determined in a third group of cells derived from a subject suffering from Parkinson's disease; if the at least one phenotype determined in the first group of cells is more similar to the at least one phenotype determined in the third group of cells than the at least one phenotype determined in the second group of cells, it indicates that the subject is at high risk of suffering from Parkinson's disease, wherein the first, second and third groups of cells are induced pluripotent stem cells, or cells differentiated from induced pluripotent stem cells, and wherein the phenotype is Miro degradation after mitochondrial depolarization. In one embodiment, the detected reaction is a change in mitochondrial function, mitochondrial fission, fusion, morphology, mitochondrial autophagy, mitochondrial transport, or other cellular features that depend on Miro function. In some cases, the comparison is performed on a computer. In some cases, the Miro level in the Parkinson's disease cells is increased compared to cells from healthy subjects. In some cases, the Parkinson's disease cells show Miro levels comparable to healthy subjects. In some cases, the mitochondrial depolarizing agent is not FCCP. In some embodiments, the Miro reducing agent is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule or an aptamer. In some cases, the Miro is detected by ELISA, Western blotting or fluorescent real-time imaging.

[0166] In one aspect, the invention provides a method for selecting a subject to receive a therapeutic agent to treat a neurodegenerative disorder, comprising:

[0167] (a) collecting cells from the subject and evaluating a first control portion of the cells for the pre-depolarization Miro level in the cells;

[0168] (b) contacting a second test portion of the cells with a depolarizing agent; and

[0169] (c) evaluating the post-depolarization Miro1 level in a second test portion of the cells contacted with the depolarizing agent, and comparing the Miro1 level to the pre-depolarization Miro1 level in a first control portion of the cells.

[0170] In some cases, the subject is treated for a neurodegenerative disorder with a therapeutic agent when the post-depolarization Miro1 level in the second test portion of the cells is reduced by 5% to 70%, 10% to 70%, 20% to 80%, 25% to 90%, 5% to 50%, or 10% to 50% relative to the pre-depolarization Miro1 level in the first control portion of the cells. In certain embodiments, the depolarizing agent reduces the post-depolarization Miro1 level in the second test portion of the cells relative to the pre-depolarization Miro1 level in the first control portion of the cells by 5% and at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%.

[0171] In some cases, the neurodegenerative disorder is Parkinson's disease. In some cases, the cell is a fibroblast. In some cases, the cell is an induced pluripotent stem cell or a cell differentiated from an induced pluripotent stem cell. In some cases, the cell is a neuron differentiated from an induced pluripotent stem cell. In some cases, the neuron is a dopaminergic neuron. In some cases, the depolarizing agent is FCCP.

[0172] The present invention relates to a method for identifying an agent for correcting a phenotype associated with Parkinson's disease or susceptibility to Parkinson's disease, the method comprising contacting a first cell population with a candidate agent, contacting a second cell population with a control agent, determining the two populations, and if the first population is closer to a normal phenotype than the second population after treatment, the candidate agent is identified as being correctable for the phenotype, wherein the phenotype is Miro cellular degradation and reduced Miro levels after mitochondrial depolarization. In some cases, the candidate agent is a Miro reducing agent. In some cases, the cells in the two populations have been treated with a mitochondrial depolarizing agent. In some cases, the cells in the two populations contain at least one endogenous allele associated with a neurodegenerative disorder or associated with susceptible neurodegeneration. In some cases, the Miro levels in the Parkinson's disease cells are increased compared to cells from healthy subjects. In some cases, the Parkinson's disease cells exhibit Miro levels comparable to those of healthy subjects. In some cases, the mitochondrial depolarizing agent is not FCCP. In certain embodiments, the Miro reducing agent is a protein or protein fragment, an antibody or antibody fragment, a peptide, a small molecule or an aptamer. In some cases, the Miro is detected by ELISA, Western blotting or fluorescent real-time imaging.

[0173] In one aspect, the present invention provides a method for screening candidate agents to identify a Miro reducing agent, comprising the steps of:

[0174] (a) Obtaining cells with insufficient Miro1 clearance;

[0175] (b) contacting a first test portion of the cells with a candidate agent while not contacting a second control portion of the cells with the candidate agent;

[0176] (c) contacting the first test portion and the second control portion of the cells subjected to step (b) with a depolarizing agent;

[0177] (d) evaluating the level of Miro1 in the first test portion relative to the second control portion.

[0178] In some cases, if the candidate agent reduces the Miro level in the first test portion relative to the Miro level in the second control portion by two or more, three or more, four or more, five or more, six or more, or seven or more standard deviations, the candidate agent is identified as a Miro reducing agent. In some cases, the first and second test portions contain cells obtained from subjects identified as having Parkinson's disease or at risk of developing Parkinson's disease. In some cases, the cells are fibroblasts. In some cases, the cells are induced pluripotent stem cells or cells differentiated from pluripotent stem cells. In some cases, the cells are neurons differentiated from pluripotent stem cells. In one aspect, the present invention provides a method for treating or preventing Parkinson's syndrome by administering to a subject a Miro reducing agent identified by the screening method described herein, the amount of the Miro reducing agent administered being an effective amount for treating or preventing Parkinson's disease.

[0179] In one aspect, the present invention provides a method for reducing the risk of drug toxicity in a human subject with Parkinson's syndrome, comprising contacting one or more cells produced by the subject with a dose of a pharmaceutical preparation, determining the toxicity of the contacted one or more differentiated cells, and prescribing or administering the agent to the subject if and only if the determination of toxicity in the contacted cells is negative. The cells may be fibroblasts from the subject, induced pluripotent cells produced by the subject, or cells differentiated from an induced pluripotent stem cell line (e.g., neurons).

[0180] In one aspect, the present invention provides a method for screening cell lines with target gene mutations to obtain candidate agents for treating neurodegenerative diseases. The method includes contacting cells from subjects with insufficient or suspected insufficient MIRO1 removal with candidate agents, observing changes or no changes in the cells, and associating changes or no changes with the ability of the agent to treat the disease. Such changes can be observed by, for example, staining to obtain intracellular MIRO levels during depolarization. The method may further include comparing the cell line or its progeny with a cell line or its progeny in which the target gene mutation is not present, the latter being a normal cell line or a cell line associated with the same target disease, but without the target gene mutation present in the first cell line. The cell may be a fibroblast from the subject, an induced pluripotent cell produced by the subject, or a cell (e.g., a neuron) differentiated from an induced pluripotent stem cell line.

[0181] The present invention also provides methods for studying the mechanism of neurodegenerative disorders (e.g., Parkinson's disease). The method includes contacting a cell line or its progeny prepared by the method described herein with an agent or condition that affects a target cell pathway, and observing changes or no changes in the cell, identifying the molecular determinants of the disorder or disease. In some cases, the target cell pathway is Miro degradation after mitochondrial depolarization. In one embodiment, the cell pathway is a change in mitochondrial function, mitochondrial fission, fusion, morphology, mitochondrial autophagy, mitochondrial transport, or other cell characteristics that depend on Miro function.

[0182] In some cases, the Miro reducing agent binds to the EF-hand domain of the Miro. In some cases, the Miro reducing agent increases intracellular calcium in the cell. In some cases, the Miro reducing agent binds to the GTPase domain of the Miro. In some cases, the Miro reducing agent binds to the nucleotide binding domain of the Miro. In some cases, the Miro reducing agent binds to the microtubule binding region of the Miro. In some cases, the Miro reducing agent binds to the Pink1 phosphorylation site of the Miro.

[0183] In some cases, the MiRo reducing agent is a calcium channel blocker. Calcium channels are protein molecules containing pores that penetrate the cell membrane or organelle, which reversibly open and close, thereby regulating the entry and exit of Ca++ ions into and out of the cell or organelle. Known calcium channels include L-type, N-type and R-type calcium channels.

[0184] In some cases, the calcium channel is an L-type channel. L-type channels are characterized by (1) a "high threshold" (for activation), that is, a strong depolarization of the cell membrane in which they are located is required to open such channels; (2) a large "single channel conductance", that is, when open, each channel allows Ca+2 ions to pass through at a relatively high rate; (3) the permeability of Ba+2 is greater than Ca+2; and, of particular note, (4) sensitivity to the efficient blockade of dihydropyridine calcium channel antagonists such as nimodipine and nifedipine (the characteristic IC50 values ​​of L channel blockade of these drugs are less than 1 μM). In most cases, the calcium "action potential" mediated by L-type channels in normal physiological environments lasts relatively long, usually not less than 100 msec. In some cases, the Miro reducing agent is an L-type channel blocker.

[0185] In a certain case, the calcium channel is an N-type channel. N-type channels are high threshold channels that are best described as dihydropyridine insensitive, but can be blocked by interacting with cone snail toxin ω-conotoxin. Qualitatively, in terms of category, the inactivation rate of N-type channels is faster than that of L-type channels. Since there is overlap between L-type and N-type channel categories in this regard, the difference in inactivation kinetics does not constitute a defining feature. In some cases, the Miro reducing agent is an N-type channel.

[0186] In a certain case, the calcium channel is an R-type channel. R-type channels can be characterized as high threshold calcium channels that are relatively resistant to blockade by dihydropyridines and ω-conotoxins. Such channels are found in a variety of neurons and are particularly abundant in cerebellar Purkinje cells. R-type channels can play a role in synaptic transmission and other processes that rely on calcium influx but are insensitive to these blockers. In some cases, the Miro reducing agent is an R-type channel.

[0187] In some cases, the Miro reducing agent can be a calcium channel antagonist that can reversibly block calcium channels. In some cases, the Miro reducing agent can play a role by non-permanently (i.e., non-covalently) binding to protein molecules that constitute such channels. In some cases, the Miro reducing agent can be a calcium channel antagonist, and its blocking degree of calcium channels exceeds the blocking degree of neurotransmitter activated channels, voltage-sensitive sodium channels and potassium channels: that is, its IC50 for calcium channels is lower than the IC50 for such neurotransmitter activated channels, sodium channels and potassium channels. In some cases, the Miro reducing agent can cross the blood-brain barrier of the subject. In some cases, the Miro reducing agent can be used to treat neurodegenerative disorders associated with excessive calcium influx into neuronal cells, including Parkinson's disease.

[0188] In some examples, the effect of Miro reducing agents on intracellular calcium levels can be monitored by organic synthetic fluorescent dyes (e.g., Fura-2, Fluo-3, Fluo-4, Indo-1, Calcium green-1, Oregon green BAPTA, Rhod-2). In some embodiments, the effect of Miro reducing agents on intracellular calcium levels can be monitored by luminescent calcium indicators based on aequorin (e.g., aequorin). In other examples, the effect of Miro reducing agents on intracellular calcium levels can be monitored by fluorescent protein-based calcium indicators (e.g., DsRed / inverse pericam, YC 2.1, G-Camp, YC 3.1, G-Camp2, Synapcam, YC 2.1, YC2.12, Camgaroo, G-CaMP2, G-CaMP2, YC 3.12, Cer TN-L15, GCamp3, etc.).

[0189] Pharmaceutical composition

[0190] In some embodiments, a pharmaceutical composition comprising an effective dose of a Miro1 reducing agent is provided, the dose being sufficient to achieve a therapeutic level of Miro1, i.e., at least 1 μM, at least 5 μM, at least 10 μM, at least 20 μM, at most about 1 mM, at most about 500 μM, at most about 250 μM, at most about 100 μM, at most about 75 μM, at most about 50 μM. The unit dose can be, for example, 1 μg / kg, 10 μg / kg, 100 μg / kg, 500 μg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg or more.

[0191] The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized foreign pharmacopoeias for use in animals, more specifically, in humans. The term "carrier" or "solvent" refers to a diluent, adjuvant, excipient or solvent administered with the Miro1 reducing agent. Such pharmaceutical carriers can be, for example, lipids, such as liposomes, such as liposome dendrimers; sterile liquids, such as saline solutions dissolved in water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, saline solutions are preferred carriers. Physiological saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If necessary, the composition may also contain a small amount of wetting agent or emulsifier or pH buffer. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. The composition can be formulated into suppositories together with traditional adhesives and carriers (e.g., triglycerides). The reducing agent can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups, such as free amino groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed with free carboxyl groups, such as free carboxyl groups derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are detailed in the following publications: Remington's Pharmaceutical Encyclopedia, EWMartin, the entire publication is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of a Miro1 reducing agent, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0192] For example, the pharmaceutical composition may also include any of a variety of stabilizers, such as antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be compounded with a variety of well-known compounds that enhance the stability of the polypeptide in vivo or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or absorption). Examples of such modifiers or compounding agents include sulfates, gluconates, citrates, and phosphates. The polypeptide in the composition may also be compounded with molecules that enhance its in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0193] For further guidance on formulations suitable for various types of administration, see the following publication: Remington's Pharmaceutical Compendium, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief review of drug delivery methods, see the following publication: Langer, Science, 249: 1527-1533 (1990).

[0194] The components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, usually at least analytical grade, and more typically at least pharmaceutical grade). In addition, compositions for in vivo use are typically sterile compositions. Where a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, in particular, any endotoxins that may be present during the synthesis or purification process.

[0195] The target pharmaceutical composition is usually sterile. Sterility can be easily achieved by filtering through a sterile filter membrane (e.g., a 0.2 μm membrane). The therapeutic composition is usually placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle. The pharmaceutical composition can be stored in a unit or multi-dose container such as a sealed ampoule or vial in the form of an aqueous solution or a lyophilized preparation for reconstitution. As an example of a lyophilized preparation, a 10-mL vial is filled with 5 ml of a sterile filtered 1% (w / v) compound aqueous solution, and the resulting mixture is lyophilized. The pharmaceutical composition containing the lyophilized Miro1 reducing agent is prepared by reconstituting the lyophilized compound, such as with antibacterial water for injection.

[0196] The pharmaceutical composition can be formulated for intravenous, oral, through implant, transmucosal, percutaneous, intramuscular, intrathecal or subcutaneous administration. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In other embodiments, the pharmaceutical composition is formulated for subcutaneous administration. The following delivery systems using many conventionally used drug carriers only represent many embodiments envisioned for use of compositions of the present invention.

[0197] The components of the pharmaceutical composition may be provided separately or mixed together in a unit dosage form, for example as a dry lyophilized powder or anhydrous concentrate. When the composition is administered by infusion, it may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

[0198] In some embodiments, the pharmaceutical composition is supplied in the form of a dry sterile lyophilized powder that can be reconstituted to a concentration suitable for administration to a subject. In some embodiments, the pharmaceutical composition is supplied in the form of a water-free concentrate. In some embodiments, the pharmaceutical composition is supplied in the form of a dry sterile lyophilized powder in a unit dose of at least 0.5 mg, at least 1 mg, at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, or at least 75 mg.

[0199] Administration method

[0200] In the subject methods, the active agent may be administered to the subject using any suitable means that is expected to reduce impairment of mitochondrial integrity and / or function, reduce any associated neurological disorders, and the like.

[0201] Therefore, the medicament can be incorporated into a variety of preparations for therapeutic administration. More specifically, the medicament of the present invention can be mixed with an appropriate pharmaceutically acceptable carrier or diluent to prepare a pharmaceutical composition, and the medicament can be prepared into a solid, semi-solid, liquid or gaseous preparation tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant and aerosol.

[0202] In pharmaceutical dosage forms, the agent may be administered in the form of its pharmaceutically acceptable salt, which may also be used alone or in appropriate combination and combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary methods and agents, not limiting methods and agents.

[0203] For oral preparations, the medicament can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, in combination with conventional additives such as lactose, mannitol, corn starch or potato starch; in combination with binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; in combination with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; in combination with lubricants such as talc or magnesium stearate; and if necessary, in combination with diluents such as buffers, wetting agents, preservatives and flavoring agents.

[0204] The agent can be formulated into an injectable preparation by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent, for example, vegetable oil or other similar oils, synthetic aliphatic acid glycerides, higher aliphatic acid esters or propylene glycol; if necessary, the compound can be used in combination with conventional additives, for example, solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0205] The medicament can be used in the form of an aerosol for administration by inhalation. The compounds of the present invention can be formulated into acceptable pressurized propellants, for example, dichlorodifluoromethane, propane, nitrogen, and the like.

[0206] In addition, the medicament can be mixed with various alkalis (e.g., emulsified alkali or water-soluble alkali) to form a suppository. The compounds of the present invention can be administered rectally in the form of a suppository. The suppository can include solvents such as cocoa butter, carbowax and polyethylene glycol, and the suppository melts at body temperature and solidifies at room temperature.

[0207] The present invention can provide unit dosage forms for oral or rectal administration, for example, syrups, elixirs and suspensions, wherein each dosage unit (e.g., a teaspoon, a tablespoon of tablets or suppositories) contains a predetermined amount of a composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration can contain the inhibitor in the form of a composition, for example, a solution dissolved in sterile water, saline or other pharmaceutically acceptable carriers.

[0208] The term "unit dosage form" as used herein refers to a physically dispersed single dosage form suitable for human and animal subjects, each unit containing a predetermined amount of the compound described in the present invention, which is mixed with a pharmaceutically acceptable diluent, carrier or solvent in an amount sufficient to produce the desired effect. The specifications of the novel unit dosage form described in the present invention depend on the specific compound used, the effect to be achieved, and the pharmacokinetic characteristics of each compound in the subject.

[0209] Other modes of administration may also be used with the present invention. For example, the medicaments of the present invention may be formulated as suppositories, and in some cases, may be formulated as aerosols and intranasal compositions. For suppositories, the vehicle composition will include conventional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories may be made from a mixture containing about 0.5% to about 10% (w / w), preferably about 1% to about 2% active ingredient.

[0210] Nasal preparations typically include a carrier that neither causes irritation to the nasal mucosa nor significantly interferes with ciliary function. The present invention may use a diluent such as water, saline or other known substances. The nasal preparation may also contain a preservative such as (but not limited to) chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the target protein by the nasal mucosa.

[0211] The medicament of the present invention can be administered as an injection. Typically, the injectable composition is prepared as a liquid solution or suspension; it can also be prepared as a solid form suitable for dissolving or suspending in a liquid carrier before injection. The preparation can also be in the form of an emulsifier or active ingredient encapsulated in a liposome carrier.

[0212] Suitable excipient solvents are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vehicle may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers or pH buffers. The actual methods for preparing such dosage forms are methods known to or obvious to those skilled in the art. See, for example, Remington's Pharmaceutical Encyclopedia, Mack Publishing Company, Easton, Pa., 17th edition, 1985; Remington: The Science and Practice of Pharmacy, A.R. Gennaro, (2000) Lippincott, Williams & Wilkins. In any case, the composition or formulation to be administered will contain an amount of the agent sufficient to achieve the desired state in the subject being treated.

[0213] Pharmaceutically acceptable excipients, such as solvents, adjuvants, carriers or diluents, are readily available to the public. In addition, pharmaceutically acceptable auxiliary agents, such as pH regulators, buffers, tonicity regulators, stabilizers, wetting agents, etc., are readily available to the public.

[0214] Route of administration

[0215] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intratumoral, subcutaneous, intradermal, topical, intravenous, rectal, nasal, oral and other parenteral routes of administration. If necessary, the routes of administration can be combined or adjusted according to the medicament and / or the expected effect. The composition can be administered in a single dose or in multiple doses.

[0216] The agent can be administered to a subject using any available conventional method and route suitable for delivering conventional drugs, including systemic or local administration routes. Typically, the administration routes encompassed by the present invention include but are not necessarily limited to enteral, parenteral or inhalation routes.

[0217] Parenteral administration routes other than administration by inhalation include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes of administration, i.e., any route of administration other than the digestive tract. Parenteral administration may be performed to achieve systemic or local administration of the medicament. When systemic delivery is desired, administration typically involves local or mucosal administration of the invasive or systemic absorption of the pharmaceutical formulation.

[0218] The agent may also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (eg, using suppositories) delivery.

[0219] Methods of administering the agent through the skin or mucous membrane include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal delivery, injection, and epidermal administration. For transdermal delivery, absorption enhancers or iontophoresis are suitable delivery methods. Iontophoresis can be achieved using commercially available "patches" that utilize electrical pulses to continuously deliver the corresponding product through unbroken skin for days or longer.

[0220] Kit

[0221] The present invention also provides reagents, devices and kits thereof for implementing one or more of the above methods. The reagents, devices and kits thereof for the subjects may vary greatly. The target reagents and devices include the reagents and devices of the above methods involving the treatment of Parkinson's disease in the subject.

[0222] The present invention provides a kit having a unit dose of active agent (e.g., an oral or injectable dose). In such a kit, in addition to the container containing the unit dose, there is also a drug instruction sheet describing the use and accompanying benefits of the drug for treating the pathological state of interest. Preferred compounds and unit doses are those described above.

[0223] In one embodiment, the kit includes a MIRO1 reducing agent and a pharmaceutically acceptable carrier. In a specific embodiment, the MIRO1 reducing agent and the pharmaceutically acceptable carrier are packaged separately. For example, the MIRO1 reducing agent can be included in the kit in a dry form, packed in a container or vial, separate from the carrier. In other embodiments, the MIRO1 reducing agent is formulated in a pharmaceutically acceptable carrier.

[0224] In certain embodiments, the kit further comprises at least one additional therapeutic agent. In a specific embodiment, the additional therapeutic agent is selected from the group consisting of levodopa, dopamine agonists, MAO-B inhibitors, amantadine, anticholinergics, PUM1 antagonists, SR protein antagonists, Parkin agonists, PINK1 agonists, 4E-BP1 agonists, Drp1 agonists, Atg1 agonists, TauS2A agonists, Rbf1 agonists, Dp antagonists, E2f1 antagonists, Polo-like kinase 2 antagonists, and Notch agonists.

[0225] In addition to the above components, the target kit will further include instructions for implementing the target diagnosis or treatment method (e.g., instructions on route of administration, dosage, dosage regimen, administration site, etc.). These instructions may be present in the target kit in various forms, one or more of which may be present in the kit. One form in which these instructions may exist is printed information on a suitable medium or substrate (e.g., a sheet or sheets of paper with information printed thereon), a kit package, a package insert, etc. Another form in which it exists is a computer-readable medium with information recorded thereon, for example, a floppy disk, a CD, etc. Another form in which these instructions may exist is a website, whereby information on a remote website can be accessed via the Internet. Any suitable device may be present in the kit.

[0226] Cross the blood-brain barrier

[0227] The blood-brain barrier restricts the absorption of many therapeutic agents by the brain and spinal cord through the systemic circulation. Molecules that cross the blood-brain barrier use two main mechanisms: free diffusion; facilitated transport. Due to the presence of the blood-brain barrier, achieving beneficial concentrations of a given therapeutic agent in the central nervous system (CNS) may require the use of a drug delivery strategy. Therapeutic agents can be delivered to the CNS by a variety of methods.

[0228] One approach relies on neurosurgical techniques. For severely ill subjects, such as accident victims or subjects with various forms of dementia, surgical intervention is indicated despite the attendant risks. For example, therapeutic agents can be delivered by direct physical introduction into the CNS, such as intraventricular or intrathecal injection of the drug. Intraventricular injection can be provided by means of an intraventricular catheter, such as one connected to a reservoir (e.g., an Ommaya reservoir). Introduction methods can also be provided by rechargeable or biodegradable devices. Another approach is to disrupt the blood-brain barrier by substances that increase the permeability of the blood-brain barrier. Examples include intra-arterial infusion of agents with poor diffusion capacity such as mannitol, drugs that increase cerebral vascular permeability such as etoposide, or vasoactive agents such as leukotrienes. Neuwelt and Rappoport, (1984) Proc. Soc. Am., 43:214-219; Baba et al., (1991) J. BFCM, 11:638-643; and Gennuso et al., (1993) Cancer Res., 11:638-643.

[0229] In addition, it may be desirable to administer the drug formulation locally to the area requiring treatment; this may be accomplished by local infusion, injection, via a catheter, or via an implant, which may be a porous, non-porous, or gel-like material, including membranes such as silicone rubber membranes or fibers, such as during surgery.

[0230] Therapeutic compounds can also be delivered by using pharmacological techniques (including chemical modification) or by screening for analogs that can cross the blood-brain barrier. The compound can be modified to increase the hydrophobicity of the molecule, to reduce the net charge or molecular weight of the molecule, or to resemble molecules that are normally transported across the blood-brain barrier. Levin, (1980) Journal of Molecular Chemistry, 23: 682-684; Pardridge, (1991) Brain delivery of peptide drugs; and Kostis et al., (1994) Journal of Clinical Pharmacology, 34: 989-996.

[0231] Encapsulating the drug in a hydrophobic environment (e.g., liposomes) can also effectively deliver the drug to the CNS. For example, WO 91 / 04014 describes a liposomal delivery system in which the drug is encapsulated within liposomes to which molecules that are normally transported across the blood-brain barrier are incorporated.

[0232] Another method of formulating a drug that crosses the blood-brain barrier is to encapsulate the drug in a cyclodextrin. Any suitable cyclodextrin that crosses the blood-brain barrier can be used, including but not limited to J-cyclodextrin, K-cyclodextrin and derivatives thereof. Generally, see U.S. Pat. Nos. 5,017,566, 5,002,935 and 4,983,586. Such compositions may also include glycerol derivatives as described in U.S. Pat. No. 5,153,179.

[0233] Delivery can also be achieved by conjugating the therapeutic agent to a transportable agent to create a new chimeric transportable therapeutic agent. For example, vasoactive intestinal peptide analog (VIPa) can only exert its vasoactive effect after conjugation to a specific carrier molecule, a monoclonal antibody (Mab) to the transferrin receptor, thereby promoting the absorption of the VI-Pa-Mab conjugate across the blood-brain barrier. Pardridge (1991); and Bickel et al., (1993) Proceedings of the National Academy of Sciences of the United States of America, 90: 2618-2622. Several other specific transport systems have been identified, including but not limited to transport systems for transporting insulin or insulin-like growth factors I and II. Other suitable non-specific carriers include but are not limited to pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives. Certain prodrugs have been described in which the drug is cleaved from the carrier upon entry into the central nervous system to release the active drug. U.S. Patent No. 5,017,566

[0234] Example

[0235] Example 1: Assay to Identify Miro Reductants

[0236] Cell culture

[0237] Human dermal primary fibroblasts isolated from PD patients (ND33424) and healthy controls (ND36091) were cultured at 37°C and 5% CO 2 The cells were cultured in a humidified air incubator in a cell culture medium DMEM (ThermoFisher, 11995-065) supplemented with 10% fetal bovine serum (Gemini Bio-Products, 900-108, heat inactivated), 1x AntiAnti (ThermoFisher, 15240096) and 1x GlutaMax (ThermoFisher, 35050061).

[0238] Western blotting

[0239] The fibroblasts were placed at 37°C and 5% CO 2 Cultured in high-glucose DMEM medium (SH30243.01, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (F0926, Sigma-Aldrich; 900-108, Gemini Bio Products) in a humidified air incubator. The medium was refreshed every 3-4 days and separated every 7-8 days. CCCP (C2759, Sigma-Aldrich) was prepared at a concentration of 40 mM in fresh DMSO each time and coated at a concentration of 40 μM in fresh medium (1:1000 dilution). IP was performed as described in (Hsieh et al., 2016).

[0240] To transfect fibroblasts, the medium was replaced with Opti-MEM (Gibco) before transfection. At room temperature (22°C), 0.5 μg DNA or 2 μl Lipofectamine 2000 was diluted in Opti-MEM in two separate tubes to a final volume of 50 μl, and the contents of both tubes were gently mixed, incubated at room temperature for 25 minutes, and then added to the fibroblasts. Six hours after transfection, the Opti-MEM containing the DNA-Lipofectamine complex was replaced with regular medium. 18 hours after transfection, fibroblasts were subjected to live imaging or treated with Miro1 reducing agent and / or CCCP.

[0241] As previously described, mitochondria were isolated from cultured human fibroblasts with slight modifications. In short, CCCP dissolved in DMSO or DMSO treated fibroblasts of the same volume were removed with a cell scraper and mechanically homogenized with a Dounce homogenizer in 750 μl separation buffer (200 mM sucrose, 10 mM TRIS / MOPS, pH 7.4). After centrifugation at 500 g for 10 minutes, the crude supernatant was centrifuged at 10,000 g for 10 minutes to precipitate complete mitochondria. The mitochondrial pellet was washed twice with separation buffer. After this step, the supernatant was called the “cytosolic fraction (Cyto)” and the pellet was resuspended in 50 μl of lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, and 1% Triton X-100–T8787, Sigma-Aldrich) containing 0.25 mM phenylmethylsulfonyl fluoride (P7626, Sigma-Aldrich) and protease inhibitors (Roche) and called the “mitochondrial fraction (Mito)”.

[0242] The sample was mixed with 2×laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl, 0.02% bromophenol blue, 700 mM 2-mercaptoethanol) at a ratio of 1:1, boiled for 5 minutes, and then loaded into SDS-PAGE chromatograph (Mito:Cyto=25:1). Electrophoresis was performed using 10% polyacrylamide gel (acrylamide:bisacrylamide=29:1) and Tris-glycine-SDS buffer (24.8 mM Tris, 192 mM glycine, 0.1% SDS).

[0243] After electrophoresis, wet transfer was performed on ice at 360 mA for 2 hours using a nitrocellulose membrane (1620115, Bio-Rad) and Tris-glycine buffer (24.8 mM Tris, 192 mM glycine). The transferred membranes were first blocked overnight at 4°C in phosphate-buffered saline (PBS) containing 5% skim milk and 0.1% tween-20, and then incubated with the following primary antibodies in blocking buffer at 4°C overnight: mouse anti-Miro1 (WH0055288M1, Sigma-Aldrich, 1:1,000), rabbit anti-Miro1 (HPA010687, Sigma-Aldrich, 1:1,000), rabbit anti-Miro2 (HPA012624, Sigma-Aldrich, 1:800), rabbit anti-VDAC (4661S, Cell Signaling Technology, 1:1,000), mouse anti-Mitofusin2 (H00009927-M01, Abnova, 1:1,000), mouse anti-Parkin (sc32282, Santa Cruz Biotechnology, 1:800), and mouse anti-VDAC (4661S, Cell Signaling Technology, 1:1,000). Biotechnology, 1:500), rabbit anti-LRRK2 (NB300-268, Novus Biologicals, 1:500), rabbit anti-OPA1 (ab42364, Abcam, 1:750), mouse anti-β-actin (A00702, Genscript, 1:1,000), mouse anti-ubiquitin (A-104, Boston Biochem, 1:500), or rabbit anti-GAPDH (5174S, Cell Signaling Technology, 1:3,000). HRP-conjugated goat anti-mouse or rabbit IgG (Jackson ImmunoResearch Laboratories) was used at a ratio of 1:5-10,000. ECL immunoblotting was performed using West DuraECL reagent (34075, GE Healthcare). The membrane was exposed to UltraCruz autoradiographic film (Santa Cruz Biotechnology) and developed on a Konica Minolta SRX-101A developer. For fluorescent western blotting, blots were probed with Cy5-conjugated goat anti-mouse IgG (PA45009, GE Healthcare) at a ratio of 1:5,000 and scanned at 635 nm using a Molecular Dynamics Storm 860 Imager (Amersham BioSciences, Piscataway, NJ) to achieve linear range fluorescence detection. Representative raw blots are listed in the Supplementary Table. Experiments were repeated 3 more times.

[0244] Statistical analysis of Western blot data of fibroblasts All experiments were performed in a blinded fashion, and the identities of the cell lines were unblinded by us (NINDS cell lines) or by PPMI investigators (PPMI cell lines). Films were scanned or digital blots were exported as 16-bit tiffs. The intensities of protein bands were measured using ImageJ (version 1.48V, NIH). The intensity of each band in the mitochondrial fraction was normalized to the intensity of the mitochondrial loading control VDAC from the same blot and expressed as a ratio to the mean of Healthy-1 treated with DMSO; this control was included in each independent experiment. MeanS values. Mean ± SEM for Miro1 are reported in Table S1A. Means for Miro1, Mitofusin2, LRRK2, and Parkin were imported into Figure 1D Heatmap shown. There was no significant difference in VDAC band intensity between all fibroblast cell lines and conditions (p = 0.8490, adjusted one-way ANOVA post hoc Tukey test). n = 3-35 independent experiments. Mann-WhitneyU test was used to compare normalized Miro1 band intensity (DMSO vs. CCCP) in the same subject, and P values ​​are given in Table S1A. The number of subjects with P values ​​> 0.05 and < 0.05 were calculated separately and used in Fisher's exact test shown in Table A. Linear regression analysis was used to determine the correlation with the Miro1 intensity ratio (average intensity of CCCP / DMSO). The band intensity at baseline was subjected to an adjusted one-way ANOVA post hoc Tukey test (P > 0.3509 for all markers in "Cyto" + "Mito"). Statistical analysis was a two-way analysis performed using Prism software (version 5.01, GraphPad).

[0245] ELISA

[0246] All experiments were performed in a blinded format. 40 μM CCCP dissolved in DMSO or the same volume of DMSO alone was applied to fibroblasts and placed for 6 hours, then the cells were lysed in a lysis buffer (100 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate) and a protease inhibitor cocktail (539134, Calbiochem). Cell debris was removed by centrifugation at 17,000 g for 10 minutes at 4°C. Microplates (MaxiSorp, NUNC) were coated with mouse anti-Miro1 (clone 4H4, WH0055288M1, Sigma-Aldrich, 1:1,000), chicken anti-β-actin (LS-C82919, life BioSciences, 1:750), mouse anti-β-actin (A00702, Genscript, 1:1000), mouse anti-ATP5β (ab14730, Abcam, 1:1000) in 0.1 M sodium carbonate-bicarbonate buffer (3:7, pH = 9.6) at room temperature overnight, while the plate cover was covered to prevent evaporation. After washing the wells in washing buffer (0.05% Tween 20 dissolved in PBS, pH 7.3), nonspecific binding sites were blocked in PBS containing 2% BSA (BP-1600-100, Fisher scientific) for 1 hour. Then, 50 μl of the cell lysate prepared above, purified full-length Miro1 protein (0-900 ng / ml, ab163047, Abcam), serial dilutions of cell lysate of fibroblasts (Healthy-1) or HEK cells (1 / 16×-2×) were added and incubated at room temperature for 2 hours. After washing, the plate was incubated with biotinylated rabbit anti-Miro1 (ARP44818_P050, Aviva Systems Biology, 1:1000) or biotinylated rabbit anti-β-actin (#5057S, Cell Signaling Technology, 1:500) in 100 μl diluent (1% BSA dissolved in PBS, pH=7.3) for 2 hours. Then, the plate was washed and incubated with horseradish peroxidase-conjugated streptavidin (21130, Thermo Scientific) at a ratio of 1:2000 in 100 μl diluent for 20 minutes. The plate was washed again, and 100 μl of tetramethylbenzidine liquid substrate (SB01, Life Technologies) was added and incubated for another 20 min. 50 μl of 1 M H 2The colorimetric reaction was terminated with SO4, and the absorbance was read at 450 nm using a microplate reader (FlexStation 3, Molecular devices). Each well plate was involved in the experiment used to generate the standard curve, and a representative standard plot is shown in the figure. Each data point is derived from 4 independent experiments (2 technical replicates each). The Mann-Whitney U test was used to compare Miro1 signals in the same subject (DMSO vs. CCCP). There was no significant difference in basal Miro1 signal between all cell lines (P>0.1177, adjusted one-way ANOVA post hoc Tukey test). The distribution of data points is represented as a box and whisker plot (extreme values, quartiles, median).

[0247] GTPase

[0248] IPed Miro1 was eluted from the protein A magnetic beads by incubation with 60 μl 0.2 M glycine (pH 2.5) for 10 min and then centrifugation at 3000 g for 2 min. The supernatant (eluate) was collected and neutralized by adding an equal volume of Tris pH 8.0. The eluate was treated with a GTPase activity kit for colorimetric analysis of GTPase activity at room temperature according to the manufacturer's instructions (602-0120, Novus Biologicals). 100 μl of the eluate was mixed with 100 μl of substrate / buffer mixture (20 μl 0.5 M Tris buffer, 5 μl 0.1 M MgCl 2 , 10 μl 10 mM GTP and 65 μl ddHO 2 O) and mixed. Prepare 200 μl of inorganic phosphate (Pi) standard (0-50 μM) in water. Next, add 50 μl of PiColorLock to the Pi standard or sample. TM mixture. After 2 minutes, stabilizer was added and mixed thoroughly. After 30 minutes, absorbance was read at 650 nm using a microplate reader (FlexStation 3, Molecular devices). The assay was validated using purified Miro1 protein (ab163047, Abcam). There was a linear correlation between the concentration of purified Miro1 protein (0-900 ng / ml) and the absorbance at 650 nm, reflecting the released Pi(R 2 =0.9711, P=0.0021). Removal of GTP or Miro1 abolished this signal (Pi<3μM). The experiment was repeated twice.

[0249] qPCR

[0250] According to the manufacturer's instructions, use (GIBCO) At least 10 6 Total RNA was extracted from 10 cells. The concentration of total RNA was measured using Nano Drop. 1 μg of total RNA was then subjected to DNA digestion using DNase I (Ambion) and immediately reverse transcribed using iScript Reverse Transcription Supermix (1708841, BIO-RAD). StepOnePlus TM Instrument (Thermo Fisher Scientific) and qPCR was performed using Green Supermix (172-5270, BIO-RAD). Human GAPDH was amplified as an internal standard. TM Expression levels were analyzed using the ELISA software (version 2.2.2). The relative expression of Miro1 was divided by the relative expression of GAPDH in the same experiment. Each sample was analyzed in 4 independent biological replicates. The following primers were used:

[0251] GAPDH forward: 5'-ACCACAGTCCATGCCATCAC-3' (SEQ ID NO: 1)

[0252] GAPDH reverse: 5'-TCCACCACCCTGTTGCTGT-3' (SEQ ID NO: 2)

[0253] Miro1 forward: 5'-GGGAGGAACCTCTTCTGGA-3' (SEQ ID NO: 3)

[0254] Miro1 reverse: 5'-ATGAAGAAAGACGTGCGGAT-3' (SEQ ID NO: 4).

[0255] Screening assay protocol

[0256] Fibroblasts were plated at a density of 2000 cells / well in clear-bottom / black-walled 384-well plates (EK-30091, E&K Scientific, Santa Clara, CA) using a Matrix Wellmate dispenser (ThermoScientific, Sunnyvale, CA) and the plates were placed at 37°C, 5% CO 2Next, chemical library compounds at defined concentrations were added using a fully automated liquid handling system, the Caliper Life Sciences Staccato system, equipped with a Twister II robot and an integrated V&P Scientific pin tool, and the well plates were placed at 37°C / 5% CO 2 Then, 20 μM FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Sigma-Aldrich, C2920) dissolved in cell culture medium was added to the specified wells, and the well plate was placed at 37°C and 5% CO 2Cells were fixed with ice-cold 90% methanol (Fisher, 482332) for 20 min at -20°C, incubated with blocking buffer (10% normal goat serum (NGS) (ThermoFisher, 50062Z), 0.5% BSA (ThermoFisher, BP1600), 0.2% Triton X-100 (ThermoFisher, T8787)) for 15 min at room temperature (RT), and then incubated with anti-Miro1 solution (Sigma-Aldrich, HPA0101687) in blocking buffer O / N at 4°C at a ratio of 1:100. The samples were washed with 1x PBS (ThermoFisher, 10010-049) using a multivalve plate washer (Bio-Tek, ELx405UV), incubated with goat anti-rabbit IgG (H+L) Cross-Adsorbed Alexa Fluor 488 (ThermoFisher, A11008) at a ratio of 1:500 at 25°C in blocking buffer for 2 hours, and washed again with 1x PBS, and finally 1.0 μg / mL DAPI (ThermoFisher, D1306) dissolved in 1x PBS was added, and the wells were sealed with PlateLoc (Velocity11, 01867.001). Unless otherwise stated, all liquids were dispensed using Multidrop 384 (Titertek, 5840200). ImageXpressMicro (Molecular Devices, IXMicro) was used to automatically image the fluorescence signal in the well plate, and MetaXpress Analysis (Molecular Devices) was used to analyze the data. The formula for calculating the percentage reduction of Miro1 is as follows: Miro1 positive cell % / median value = [(positive cell Miro integral intensity / all cell nucleus Miro integral intensity)-the well plate median value calculated in the same way (the median value % of positive cell Miro / cell nucleus Miro in the well plate data)] / [the well plate median value calculated in the same way (the median value % of positive cell Miro / cell nucleus Miro in the well plate data)]. Positive cells are cells with Miro1 immunostaining. Zero value means no difference (inactivation) with the median value. Negative value means that Miro is reduced. Positive value means that Miro is increased. Screening and data analysis were performed together with the High Throughput Bioscience Center (HTBC) of Stanford University.

[0257] Hit compound confirmation - Immunocytochemistry, confocal microscopy, image analysis

[0258] Fibroblasts were plated at a density of 400,000 cells / well in a 6-well plate (VWR, 10861-554) on a cover glass (Fisher scientific, 22-293232) and the plate was placed at 37°C, 5% CO 2 Then, the newly ordered compounds dissolved in DMSO were added at the specified concentrations and the plates were placed at 37°C / 5% CO 2 Then, FCCP (final concentration 20 μM, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Sigma-Aldrich, C2920) dissolved in cell culture medium was added to the specified wells, and the well plate was placed at 37°C and 5% CO 2 The cells were fixed with ice-cold 90% methanol (Fisher, 482332) for 20 minutes at -20°C, incubated with blocking buffer (10% normal goat serum (NGS) (ThermoFisher, 50062Z), 0.5% BSA (ThermoFisher, BP1600), 0.2% Triton X-100 (ThermoFisher, T8787)) for 15 minutes at 25°C, and then incubated with anti-Miro1 solution (Sigma-Aldrich, HPA0101687) at a ratio of 1:100 at 4°C in blocking buffer. The samples were washed three times with 1x PBS (ThermoFisher, 10010-049), incubated with goat anti-rabbit IgG (H+L) Cross-Adsorbed Alexa Fluor 488 (ThermoFisher, A11008) at a ratio of 1:500 at 25°C in blocking buffer for 2 h, and washed three times with 1x PBS again. Finally, the samples were incubated with NucBlue TM ProLong stain TMGlass AntifadeMountant (Sclerostin, Thermofisher, P36983) was mounted on slides and allowed to cure O / N. Samples were imaged on a Leica SPE laser scanning confocal microscope (JH Technologies) at 25°C using a 20× / NA0.60 oil Plan-Apochromat objective with identical imaging parameters between genotypes. Images were processed using ImageJ (version 1.48, NIH) using the IntensityRatio Nuclei Cytoplasm tool (http: / / dev.mri.cnrs.fr / projects / imagej-macros / wiki / Intensity_Ratio_Nuclei_Cytoplasm_Tool) and plotted for mean cytoplasmic intensity (average intensity of the cytoplasmic area).

[0259] Drugs from the NIH clinical sample library were screened at a defined concentration of 10 μM, with four biological replicates for each drug (377 compounds). Eleven of the 377 unique compounds were observed to reduce Miro1 protein levels by three standard deviations after FCCP treatment across all biological replicates. These compounds were ranked in order of the extent of Miro1 reduction (Table 1A). The 12 small molecules that reduced Miro1 protein levels in 3 / 4 biological replicates were ranked in order of the extent of Miro1 reduction. The BioMolFDA library was screened at different 5-fold doses (1.25-2.5-5-10-20 μM) in two replicates, and three hit compounds were identified that partially but significantly reduced Miro1 protein levels in a dose-dependent manner after FCCP treatment. These hit compounds were ranked in order of the extent of Miro1 reduction, and six compounds were found to reduce Miro1 protein levels by 20% or more (Table 1A). In addition, another 12 small molecules reduced Miro1 protein levels in 3 / 4 biological replicates (Table 1B). Next, we screened the BioMol FDA library with 5-fold doses (1.25-2.5-5-10-20 μM) and repeated twice. Among the 175 unique compounds, we identified 3 hit compounds that partially but significantly reduced Miro1 protein levels in a dose-dependent manner after FCCP treatment. We ranked these 3 hit compounds according to the degree of Miro1 reduction (Table 1A). Overall, we identified 14 active substances (primary hit rate 2.5%) and 12 potential active substances.

[0260] To reduce experimental bias (e.g., freshness of compounds, experimental reagents, and imaging equipment), 14 hit compounds identified in the high-throughput screen were individually validated. All 14 compounds were tested at the highest screening concentration using the same sporadic PD cell line with at least 4 biological replicates. Miro1 levels were evaluated in the absence of mitochondrial depolarization (DMSO) but with compound treatment, which revealed the drug effect of Miro1 on polarizing healthy mitochondria. Samples were imaged under a confocal microscope with identical settings, and negative controls without primary antibody produced no signal under the imaging settings. Seven of the initial 14 hit compounds were found to consistently reduce Miro1 protein after FCCP treatment (Table 1A). One compound (i.e., fenbufen) appeared to be toxic (Table 1A). Among the seven confirmed hit compounds, the compounds tranilast and benidipine hydrochloride had no effect on basal Miro1 levels, while the compounds physostigmine, pravastatin sodium, lofepramine, temozolomide, and escitalopram oxalate reduced Miro1 protein at baseline.

[0261] Example 2: Miro1 marks Parkinson's disease subpopulation

[0262] Identification of molecular targets and pharmacodynamic markers for Parkinson's disease (PD) will enable more effective clinical management and experimental treatments. Miro1 is located on the mitochondrial surface and mediates mitochondrial motility. Miro1 is removed from depolarized mitochondria to facilitate its clearance (clearance by mitophagy). In this study, we explored the clinical utility of Miro1 in detecting PD and evaluating potential treatments. We measured the response of Miro1 to mitochondrial depolarization in skin fibroblasts from a number of PD subjects using a biochemical assay and found that fibroblast cell lines from more than 94% of the subjects were unable to remove Miro1 after depolarization.

[0263] Miro1 is resistant to depolarizing mitochondrial removal in skin fibroblasts from a large cohort of PD subjects. Skin fibroblasts can be easily obtained from subjects by a minimally invasive, painless procedure. We aimed to determine the frequency of the Miro1 phenotype in skin fibroblasts from a large cohort of sporadic and familial PD subjects. We fractionated mitochondria after CCCP treatment, which depolarizes the mitochondrial membrane potential (ΔΨm). In wild-type controls 6 h after treatment, Miro1 and Mitofusin2 were removed from damaged mitochondria, as detected by Western blotting ( Figure 1A , 1B).

[0264] Table S1A. Related to Figure 1. Demographic information and Miro1 values. Demographic information and clinical scores were obtained from the consortium's online database. Not all information was available for all subjects. Specific analyses used data from subjects with available information. P values ​​were derived by comparing normalized Miro1 intensity within the same subject (CCCP vs. DMSO).

[0265] The OMM protein Mitofusin2 is a target of the PINK1-Parkin pathway, but not of LRRK2, for depolarization-triggered degradation. We included Mitofusin2 in our readout to compare its phenotypic frequency in PD and Miro1. We screened a total of 71 PD and 3 high-risk fibroblast cell lines, including the entire PD fibroblast bank of the National Institute of Neurological Disorders and Stroke (NINDS) Human and Cell Repository and the first PD control cohort published by the Parkinson's Progression Markers Initiative (PPMI). All subjects had a diagnosis of PD and no other signs of neurological disease. High-risk subjects were younger asymptomatic family members of the proband and carried the same genetic mutation (mutation in LRRK2 or SNCA). Twenty-eight subjects had a positive family history. We included 22 controls, including 12 age / sex / race-matched healthy subjects recruited from the same cohort and 10 subjects with other neurological diseases, including Huntington's disease (HD) or Alzheimer's disease (AD) (Table A). We performed the assay in a blinded manner.

[0266] Table A

[0267] Summary of Miro1 phenotypes for all subjects used in this study.

[0268]

[0269] P values ​​were determined using Fisher's exact test compared to PD. In ELISA or western blotting, Miro1 intensity after DMSO and CCCP treatment in the same subject was compared by Mann-Whitney test, and the number of subjects with P>0.05 or <0.05 was defined as "number (Miro1 DMSO vs. CCCP P>0.05 or <0.05)".

[0270] Notably, we found a consistent impairment in the removal of Miro1 from mitochondrial fractions following CCCP treatment for 6 h in 69 PD and high-risk cell lines (93.2%). In contrast, Miro1 was efficiently removed following depolarization in every single control subject (0%) (Table A). This phenotype was more clearly demonstrated when we imported the average band intensities into a heatmap, where the absence of color change following treatment indicated a failure to remove Miro1 ( Figure 1D). Miro1 achromaticity is common in PD subjects. After CCCP treatment, a small number of PD cell lines also failed to remove Mitofusin2 ( Figure 1D The basal protein levels of Miro1 and Mitofusin2 were similar in all cell lines ( Figure 1D ; P>0.0906).

[0271] This phenotype in Miro1 depletion was significantly associated with PD (P<0.00001). The ratio of Miro1 intensity (CCCP treatment / DMSO treatment) was also significantly associated with PD (P<0.0001; Figure 2 A), but not age (at sampling and onset) or sex ( Figure 2 B-2D). After receiving the Unified Parkinson's Disease Rating Scale (UPDRS), Hoehn and Yahr Scale, or Mini-Mental State Examination ( Figure 2 In subjects with PD (Figure 2E-2H), there was no clear correlation between Miro1 ratio and disease progression (years with PD) or clinical manifestations. We demonstrated that cell passage number in the range of 5-19 had no effect on this phenotype. Overall, these observations suggest that the inability to remove Miro1 from damaged mitochondria is a common cellular defect in a large population of PD subjects.

[0272] The LRRK2 and PINK-Parkin pathways are extensively affected in PD fibroblasts. We have previously identified two parallel molecular pathways that are both essential for the removal of Miro1 from the OMM of depolarized mitochondria (the LRRK2 and PINK1-Parkin axes). To investigate the mechanisms underlying the accumulation of Miro1 on damaged mitochondria in PD fibroblasts, we tested the hypothesis that this accumulation is due to impairment of the LRRK2 or PINK1-Parkin pathways. We have established that in wild-type control fibroblasts, mitochondrial depolarization achieved by CCCP treatment for 1 h triggers the recruitment of cytosolic LRRK2 and Parkin to mitochondria and Miro1, followed by the removal of Miro1 at 6 h ( Figure 1A , 1C). Antibodies against LRRK2, Parkin, and Miro1 have been validated in human cells lacking the corresponding genes.

[0273] We screened all 96 of these fibroblast cell lines using this readout and found a variety of phenotypes in the cells from our subjects. Some lines were impaired only in recruiting LRRK2 to depolarized mitochondria, some were impaired only in recruiting Parkin, and some were defective in recruiting both proteins. Since the inability to relocalize LRRK2 to damaged mitochondria interferes with the subsequent removal of Miro1, but not Mitofusin2, this result could explain the lower frequency of the Mitofusin2 phenotype compared to Miro1 in these subjects ( Figure 1D We found that in seven PD cell lines, the recruitment of LRRK2 and Parkin to damaged mitochondria appeared normal compared with controls, whereas Miro1 removal was still affected ( Figure 1D ), suggesting that other mechanisms may be at work in these cells. Basal levels of LRRK2 and Parkin were comparable in all cell lines (P>0.8684). In conclusion, our results suggest that LRRK2 and PINK1-Parkin pathways are largely affected in fibroblasts from PD subjects, leading to downstream convergence and failure to remove Miro1 from damaged mitochondria.

[0274] ELISA confirmed the high frequency and specificity of the Miro1 phenotype in PD fibroblasts. We established an enzyme-linked immunosorbent assay (ELISA) to detect Miro1 in response to CCCP ( Figure 3 ), which can be used for clinical laboratory purposes. We examined Figure 1D 14 PD / high-risk cell lines and 15 control cell lines were used in the study. For each cell line, the response of Miro1 to mitochondrial depolarization was analyzed ( Figure 3 A) is consistent with the results obtained using mitochondrial fractionation and Western blotting ( Figure 1D , Table S1A). We used this ELISA to validate other independent cohorts. We included 40 healthy controls and 12 PD subjects from the Stanford Alzheimer's Disease Research Center (ADRC) and the Coriell Institute (Table S1B). We found that Miro1 was efficiently degraded after depolarization in all control cell lines, but not in any PD subjects ( Figure 1E); this finding validates the reliability of high-frequency Miro1 accumulation on depolarized mitochondria in PD populations (Fig. 1, Table A). We also obtained cell lines from subjects with movement disorders that exhibited clinical manifestations similar to PD, including 4 cases of sporadic dementia with Lewy bodies (DLB; Stanford), 3 cases of frontotemporal lobar degeneration (FTD; Coriell and NINDS), 2 cases of sporadic corticobasal degeneration (CBD; Mayo Clinic), and 3 cases of sporadic progressive supranuclear palsy (PSP; Mayo Clinic) (Table S1B). In all cell lines, Miro1 was effectively degraded after CCCP treatment ( Figure 1F ), indicating that Miro1 accumulation is specific to PD. Establishing an ELISA to detect Miro1 will help the clinical application of our findings.

[0275] Example 3: Effect of Miro reductant on mitochondrial motility

[0276] Dopaminergic neurons expressing LRRK2G2019 were grown on glass coverslips and placed in 35 mm dishes containing Hibernate E low fluorescence medium (BrainBits) on a 37°C heating stage and imaged using a 63× / NA0.9 water immersion objective with an excitation wavelength of 561 nm or 488 nm. Neurons expressing LRRK2GS2019 were divided into test and control neuron pools. The test pool was treated with the Miro reducing agent identified in Example 1, while the control pool was not treated. After 10 hours, both neuron pools were treated with FCCP for 14 hours. Mitochondrial motility was measured.

[0277] Time-lapse images were acquired continuously at 3-5 second intervals before and after the addition of Miro reducing agent (10 μM). Axons longer than 50 μm were selected for recording. Image recording time was 120 to 300 minutes. When necessary, 250 nM TMRM was applied and left for 30 minutes. For quantification, kymograms were generated from time-lapse images using ImageJ, representing 100 second time periods at different time points before or after the addition of antimycin A. Each kymogram was then imported into a macro written in Labview (NI, TX), and a mouse-driven cursor was used to track a single mito-dsRed dot in the center of the mito-dsRed object. Matlab (MA thworks, MA) was used to quantify the following parameters: 1) the instantaneous velocity of each mitochondria, 2) the average velocity of mitochondria in motion, 3) the percentage of motion time for each mitochondria, 4) the stop frequency, and 5) the return frequency.

[0278] The length and intensity of mitochondria were measured using ImageJ. According to the above parameter measurements, neurons in the test pool with depolarized mitochondria showed reduced mitochondrial motility, while neurons in the control pool with depolarized mitochondria showed mitochondrial motility comparable to that before the addition of the Miro reducing agent.

[0279] Example 4: Effect of Miro reductant on Parkinson's disease iPSC neurons

[0280] LRRK2G2019S iPSC-derived neuronal cultures were treated with a range of concentrations of the mitochondrial stressor antimycin A for 6 h to induce oxidative stress. LRRK2G2019S iPSC-derived neuronal cultures exhibited antimycin A dose-dependent neuronal loss, indicating vulnerability to mitochondrial stress. LRRK2G2019S neurons treated with Miro-reducing agent protected against LRRK2G2019S neuronal loss caused by 1 or 10 μM antimycin A, indicating its protective effect against neuronal loss caused by mitochondrial stress.

[0281] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that the embodiments are provided by way of example only. Many variations, changes, and alternatives may now be envisioned by those skilled in the art without departing from the present invention. It should be understood that a variety of alternatives to the embodiments of the present invention described herein may be employed in the implementation of the present invention. The following claims are intended to define the scope of the present invention and accordingly encompass methods and structures within the scope of these claims and their equivalents.

[0282] Table 1A:

[0283]

[0284] A = Miro level decreased by > 30%; B = Miro level decreased by > 20% and < 30%; C = Miro decreased by > 10% and < 20%; D = Miro decreased by < 10%

[0285] Table 1B: Other small molecules that reduce Miro1 protein levels

[0286]

[0287] A = Miro level decreased by > 30%; B = Miro level decreased by > 20% and < 30%; C = Miro decreased by > 10% and < 20%; D = Miro decreased by < 10% .

Claims

1. A method for the activity screening of candidate agents for reducing MIRO1 levels, the method comprising: (a) contacting a candidate agent with a sample from a subject with insufficient or suspected insufficient MIRO1 removal; (b) measuring the MIRO1 level in the sample; (c) comparing the MIRO1 level in the sample with a control MIRO1 level; and (d) evaluating the activity of the candidate agent if the MIRO1 level in the sample is lower than the control MIRO1 level.

2. The method according to claim 1, wherein the sample comprises cells.

3. The method according to claim 1 or 2, wherein the sample comprises skin cells.

4. The method according to any one of claims 1 to 3, wherein the sample with the control MIRO1 level is from a healthy subject.

5. The method according to any one of claims 1 to 4, wherein the evaluation comprises a biochemical assay, Western blotting or ELISA.

6. The method according to any one of claims 1 to 5, wherein the MIRO1 level is about 20% or more lower than the control MIRO1 level.

7. The method according to any one of claims 1 to 6, wherein the MIRO1 level is about 30% or more lower than the control MIRO1 level.

8. A method for determining the MIRO1 status of a subject, which comprises measuring the response of Miro1 to mitochondrial depolarization using a biochemical assay, Western blotting or ELISA to determine whether there is insufficient MIRO1 removal in the subject after depolarization, and selecting a subject with insufficient MIRO1 removal after depolarization to receive treatment with an MIRO1 reducing agent.

9. The method according to claim 8, which comprises detecting the MIRO1 level in the subject and comparing the MIRO1 level with a control MIRO1 level from a control subject.

10. The method according to claim 8 or 9, wherein the detection of the MIRO1 level comprises ELISA.

11. The method according to any one of claims 8 to 10, wherein the detection of the MIRO1 level comprises detecting MIRO1 in a sample of the subject.

12. The method according to claim 11, wherein the sample comprises skin cells.

13. The method according to any one of claims 8 to 12, wherein the MIRO1 level in the subject is higher than the control MIRO1 level from a control subject.

14. The method according to any one of claims 8 to 13, wherein the MIRO1 level in the subject is about 20% or more higher than the control MIRO1 level from a control subject.

15. The method according to any one of claims 8 to 14, wherein the MIRO1 level in the subject is about 30% or more higher than the control MIRO1 level from a control subject.

16. The method according to any one of claims 13 to 15, which comprises diagnosing that the subject has a neurodegenerative disorder.

17. The method according to claim 16, wherein the neurodegenerative disorder is Parkinson's disease.

18. The method according to any one of claims 8 to 17, which further comprises treating the subject with the MIRO1 reducing agent.

19. The method according to claim 18, which further comprises monitoring MIRO1 levels before and after treatment with the MIRO1 reducing agent.

20. A method of treating a neurodegenerative disorder, which comprises administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro in sporadic Parkinson's disease cells having depolarized mitochondria by three standard deviations or more compared to the reduction of Miro in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria that have not been contacted with the Miro reducing agent.

21. The method according to claim 20, wherein the healthy cells having depolarized mitochondria are healthy cells contacted with a mitochondrial depolarizing agent.

22. A method of treating a neurodegenerative disorder, which comprises administering a Miro reducing agent to a subject in need thereof, wherein the Miro reducing agent reduces Miro in Parkinson's disease cells having depolarized mitochondria by three standard deviations or more compared to the reduction of Miro in control depolarized sporadic Parkinson's disease cells having depolarized mitochondria that have not been contacted with the Miro reducing agent.

23. A method of treating a neurodegenerative disorder, which comprises administering a Miro reducing agent to a subject in need thereof, wherein: (a) the Miro reducing agent reduces Miro in Parkinson's disease cells having depolarized mitochondria by two standard deviations or more compared to the reduction of Miro in control Parkinson's disease cells having depolarized mitochondria that have not been contacted with the Miro reducing agent; and (b) the Miro 1 reducing agent reduces Miro in Parkinson's disease cells having non-depolarized mitochondria by one standard deviation or less compared to the reduction of Miro in control Parkinson's disease cells having non-depolarized mitochondria that have not been contacted with the Miro reducing agent.

24. The method according to any one of claims 20 to 24, wherein the Parkinson's disease cells having depolarized mitochondria are Parkinson's disease cells contacted with a mitochondrial depolarizing agent.

25. The method according to any one of claims 20 to 25, wherein the Parkinson's disease cells are sporadic Parkinson's disease cells.

26. The method according to any one of claims 20 to 26, wherein the Parkinson's disease cells are fibroblasts.

27. A method of treating a neurodegenerative disorder, which comprises administering a Miro reducing agent to a subject in need thereof, wherein in the following assay, the Miro reducing agent causes a reduction in Miro: (a) inoculating fibroblasts from a sporadic Parkinson's disease patient into the wells of an array; (b) Twenty-four hours after step (a) is completed, add the candidate agent to the test wells, and do not add the candidate agent to the control wells; (c) Ten hours after step (b) is completed, add FCCP to the test wells and the control wells; (d) Fourteen hours after step (c) is completed, fix the cells in the test wells and the control wells with ice-cold 90% methanol; and (e) Immunostain the cells in the test wells and the control wells with anti-Miro and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and image with a confocal microscope to measure the Miro intensity / cell for the images of the test wells and the control wells; wherein when the candidate agent reduces Miro in the test wells by three standard deviations or more relative to the control wells, the candidate agent is a Miro reductant.

28. A method for treating a neurodegenerative disorder, comprising administering a Miro reductant to a subject in need thereof, wherein the Miro reductant reduces Miro in sporadic Parkinson's disease cells as determined by the following: (a) Seed fibroblasts from a patient with sporadic Parkinson's disease into the wells of an array; (b) Twenty-four hours after step (a) is completed, add the candidate agent to the first test well, and do not add the candidate agent to the first control well; (c) Ten hours after step (b) is completed, add FCCP to the first test well and the first control well; (d) Fourteen hours after step (c) is completed, fix the cells in the first test well and the first control well with ice-cold 90% methanol; and (e) Immunostain the cells in the first test well and the first control well with anti-Miro and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and image with a confocal microscope to measure the Miro intensity / cell for the images of the first test well and the first control well; and wherein the Miro reductant does not reduce Miro in sporadic Parkinson's disease cells as determined by the following: (a1) Seed fibroblasts from a patient with sporadic Parkinson's disease into the wells of an array; (b1) Twenty-four hours after step (a1) is completed, add the candidate agent to the second test well, and do not add the candidate agent to the second control well; (c1) Fourteen hours after step (b1) is completed, fix the cells in the second test well and the second control well with ice-cold 90% methanol; and (d1) Immunostain the cells in the second test well and the second control well with anti-Miro and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and image with a confocal microscope to measure the Miro intensity / cell for the images of the second test well and the second control well; wherein when the candidate agent reduces Miro in the first test well by two standard deviations or more relative to the first control well, and the candidate agent reduces Miro in the second test well by less than one standard deviation relative to the second control well, the candidate agent is a Miro reductant.

29. The method according to any one of claims 20 to 28, wherein the Miro reducing agent reduces the intracellular calcium level by 20% or more.

30. The method according to any one of claims 20 to 29, wherein the molecular weight of the Miro reducing agent is from 100 to 2000 Daltons.

31. The method according to any one of claims 20 to 30, wherein the Miro reducing agent is an antibody, a peptide, a protein, or a part of one of them.

32. The method according to any one of claims 20 to 31, wherein the Miro reducing agent binds to an EF hand protein.

33. The method according to any one of claims 20 to 31, wherein the Miro reducing agent is a calcium channel blocker.

34. The method according to claim 33, wherein the calcium channel blocker is an L-type and an N-type calcium channel blocker.

35. The method according to any one of claims 20 to 34, wherein the subject shows an increase in Miro.

36. The method according to any one of claims 20 to 35, wherein MIRO1 in the subject is at least about 20% or more or 30% or more higher than that in a control subject.

37. The method according to any one of claims 20 to 36, wherein the subject has no symptoms of a neurodegenerative disorder in other aspects.

38. The method according to any one of claims 20 to 37, wherein the neurodegenerative disorder is Parkinson's disease.

39. A method for selecting a subject to receive a therapeutic agent for treating a neurodegenerative disorder, comprising: (a) collecting cells from the subject and evaluating a first control portion of the cells for the pre-depolarization Miro level in the cells; (b) contacting a second test portion of the cells with a depolarizing agent; and (c) evaluating the post-depolarization Miro level in the second test portion of the cells contacted with the depolarizing agent and comparing the Miro level with the pre-depolarization Miro level in the first control portion of the cells; wherein when the post-depolarization Miro level in the second test portion of the cells is reduced by 40% or less relative to the pre-depolarization Miro level in the first control portion of the cells, the subject's neurodegenerative disorder is treated with a therapeutic agent.

40. The method according to claim 39, wherein when the post-depolarization Miro level in the second test portion of the cells is reduced by 10% to 50% relative to the pre-depolarization Miro level in the first control portion of the cells, the subject's neurodegenerative disorder is treated with a therapeutic agent.

41. The method according to claim 39 or 40, wherein the neurodegenerative disorder is Parkinson's disease.

42. The method according to any one of claims 39 to 41, wherein the subject has no symptoms of Parkinson's disease.

43. The method according to any one of claims 39 to 42, wherein the therapeutic agent is selected from levodopa and dopamine antagonists.

44. The method according to any one of claims 39 to 43, wherein the therapeutic agent is a Miro reducing agent.

45. The method according to any one of claims 39 to 44, wherein the molecular weight of the therapeutic agent is from 100 to 2000 Daltons.

46. The method according to any one of claims 39 to 44, wherein the therapeutic agent is an antibody, a peptide, or a protein or a part of one of them.

47. The method according to any one of claims 39 to 46, wherein the therapeutic agent binds to an EF hand protein.

48. The method according to any one of claims 39 to 47, wherein the therapeutic agent is a calcium channel blocker.

49. The method according to claim 48, wherein the calcium channel blocker is an L-type and an N-type calcium channel blocker.

50. A method of ameliorating an aging condition, comprising administering a Miro reducing agent to a subject in need thereof.

51. The method according to claim 50, wherein the aging condition is selected from memory impairment, muscle degeneration, arthritis, cardiovascular disease, osteoporosis, glaucoma, dementia, macular degeneration, and cataract.

52. The method according to claim 50 or 51, wherein the Miro reducing agent is administered prophylactically.

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